Redescription of Chirocephalus bobrinskii (Alcock, 1898) (Anostraca: Chirocephalidae) with special attention to somatic sensillar armature

Abstract

Systematics within the Chirocephalidae (Branchiopoda) remain confused at the genus level and in several groups within Chirocephalus. We redescribe Chirocephalus bobrinskii (Alcock, 1898), an endemic of Central Asia, based on material from several localities in Tajik Pamir, Tajikistan. The studied populations displayed significant variability of the second antenna distal antennomere, shape and size of the antennal appendage, and thoracopods, but only slight interpopulation genetic variation of the Folmer’s COI gene fragment. With such high morphological plasticity, no clear quantifiable differences were observed between C. bobrinskii and the closely related Central Asian taxa, C. altaicus Daday, 1910 and C. turkestanicus Daday, 1910, which we treat as junior synonyms. Based on morphological and genetic evidence, C. bobrinskii likely belongs to the C. spinicaudatus group, but further integrative revision of that group is needed. We also discuss the structure and location of somatic sensillae in C. bobrinskii, which differ between sexes. As these sensory structures are present in many anostracans and may have specific patterns in different groups, they may be useful in systematics. Unfortunately, the somatic sensillae are rarely included in species descriptions due to their small size. Thus, more studies are needed to clarify the significance of sensillar morphology to anostracan taxonomy.

Keywords:
Barcoding; branchiopods; Central Asia; Pamir; sensilla; temporary water bodies; variability

INTRODUCTION

Fairy shrimps (Anostraca Sars, 1867) are a large and diverse group of branchiopod crustaceans (Branchiopoda) comprising ca. 350 valid living species, which are mostly found in seasonally astatic water bodies around the world (Rogers, 2013; 2024; Marrone et al., 2017). Some anostracan taxa, namely Artemia salina (Linnaeus, 1758), are economically important (Browne et al., 1990). In recent years, anostracan faunistic diversity and phylogeography have been the subject of intense studies (e.g., Daniels et al., 2004; Rogers and Padhye, 2014; Lindholm et al., 2016; Rogers et al., 2019; Rogers and Aguilar, 2020; Shu et al., 2018; Deng et al., 2021; Boumendjel et al., 2018; 2024).

Nevertheless, the systematics of the Chirocephalidae still remain unclear (Marrone et al., 2017; Rogers, 2013; 2024). Separation of Anostraca genera is based primarily on genital morphology (Rogers, 2013; 2024 and references cited within). Anostracan species are morphologically defined by the form of the male antenna II and the morphology of female abdomen and brood pouch (e.g., Cottarelli et al., 2010; Rogers and Soufi, 2013; Rogers and Padhye, 2014; Shu et al., 2018; Boumendjel et al., 2018; 2024; Rogers et al., 2019; Rogers, 2013; 2024). Both generic and specific subdivisions based on morphological characters mentioned above have been validated by molecular analysis of several nuclear (28S D1-D3 rDNA, 18S rDNA) and mitochondrial (16S rDNA, COI) gene fragments (see Weekers et al., 2002; Remigio and Hebert, 2000 for genera and Daniels et al., 2004; Rogers and Aguillar, 2020; Asem et al., 2023; Islam et al., 2024 for species molecular discrimination). In some anostracan groups the gonopod, cercopod and resting egg morphology may also have species specific characters (Mura, 1992; 2001; Timms et al., 2004; Rogers and Ferreira, 2007; Miličić and Petrov, 2009; Rogers and Padhye, 2014; Shu et al., 2018). However, resting egg morphology can strongly vary within a particular species, thus raising questions about the value of this character for certain genera (Mura, 2001; Gadiadullina et al., 2021). In contrast, the number and location of sensory elements are usually ignored when describing a new species, and the potential of their morphology for species discrimination is unknown.

Chirocephalus Prévost, 1803 is the second largest anostracan genus, comprising at least 55 valid or potentially valid species (Rogers, 2013; 2024; Boumendjel et al., 2024) occurring in the Palearctic and Indonesia (Belk and Brtek, 1995; Mura, 2001; Rogers et al., 2019). Many Chirocephalus species are known from a single locality or have a restricted distribution range (Belk and Brtek, 1995). For instance, a number of species are known only from Central Asia, e.g. C. bobrinskii (Alcock, 1898), C. jaxartensis (Smirnov, 1948), C. turkestanicusDaday, 1910, C. tereki Brtek, 1984, and C. povolnyi Brtek, 1967 (Rogers et al., 2019).

Chirocephalus bobrinskii is one of the most widely distributed species among Central Asian endemics. It was first described from Lake Chakmaktin, Afghan Pamir as Branchipus (Chirocephalus) bobrinskii by Alcock (1898); however, the description is brief with only two images, and does not meet modern standards. Daday (1910) later elevated Chirocephalus to full genus level and described C. altaicus Daday, 1910 from the Altai Mountains (Kyrgyzstan and Russia); although C. altaicus was subsequently synonymized with C. bobrinskii (Kemp, 1911). Unfortunately, Kemp (1911) did not provide illustrations to support his conclusions. Daday (1910) described another species from South Kyrgyzstan, C. turkestanicus, which differs from C. altaicus and C. bobrinskii mostly in the shape of male second antenna (Daday, 1910; Rogers et al., 2019). Later, Vekhoff (1992) emended the description of C. altaicus based on material from Lake Maraldy in northeastern Kazakhstan. However, he considered C. altaicus as a separate species without any discussion on the relationships of this taxon and C. bobrinskii. Mura (2001) first described the resting egg morphology of C. bobrinskii and most of its congeners. Finally, Dadykin et al. (2024) reported C. cf. bobrinskii in small water bodies of the Pamir mountains, Tajikistan and briefly described the morphology of those populations. The authors also noted the presence of males resembling C. turkestanicus in the same water bodies.

Here, we revise Chirocephalus bobrinskii based on the material from Tajik Pamir, Kyrgyzstan and South Kazakhstan and discuss its taxonomic position within the genus Chirocephalus.

MATERIAL AND METHODS

Field studies, morphological analysis and images

New material was collected in June 2023 in Tajik Pamir, Tajikistan by I.A. Dadykin (Tab. 1). The collection method is described in detail by Dadykin et al. (2024). After a primary identification, the samples were stored at 4 °C. We also examined material deposited at the Zoological Museum of Russian Academy of Sciences, Saint Petersburg, Russia (see Comparative material section in results). Unfortunately, the type materials at the Indian Museum, Calcutta, India (IM) were not examined, as the Indian Museum has not responded to any requests (email, telephone calls, or personal visits) to borrow or view the specimens. We can only suspect that the types are lost.

Table 1.
List of studied specimens with information on localities and collection numbers.

Species	Studied specimens	Latitude	Longitude	Water body	Region and country	Reference	Collection numbers
Chirocephalus bobrinskii (Alcock, 1898)	2 ♂, 1 ♀	37.76927	72.99949	puddle in the valley of Alichur River, 5 km east of Lake Yashilkul	Badakhshan MAR, Tajikistan	Dadykin et al. (2024)	AAK M-8565, ZIN 55620, MGU MD-1426
C. bobrinskii	2 ♂, 1 ♀	37.76931	73.00127	puddle in the valley of Alichur River, 5 km east of Lake Yashilkul	Badakhshan MAR, Tajikistan	Dadykin et al. (2024)	AAK M-8566, MGU MD-1427
C. bobrinskii	6 ♂, 4 ♀	37.45477	73.57547	puddle on the northwest coast of Lake Zorkul	Badakhshan MAR, Tajikistan	Dadykin et al. (2024)	AAK M-8568, MGU MD-1428, MGU MD-1434
C. bobrinskii	3 ♂, 2 ♀	37.4536	73.81543	lake 3 km east of Lake Zorkul	Badakhshan MAR, Tajikistan	Dadykin et al. (2024)	AAK M-8569, MGU MD-1429
C. bobrinskii	3 ♂, 3 ♀	37.45701	73.83064	drying lake 4 km east of Lake Zorkul	Badakhshan MAR, Tajikistan	Dadykin et al. (2024)	AAK M-8570, MGU MD-1430
C. bobrinskii	8 ♂, 5 ♀	37.46001	73.83961	small lake 4 km east of Lake Zorkul	Badakhshan MAR, Tajikistan	Dadykin et al. (2024)	AAK M-8571, MGU MD-1431
C. bobrinskii	2 ♂, 3 ♀	37.45086	73.85398	large lake 5 km east of Lake Zorkul	Badakhshan MAR, Tajikistan	Dadykin et al. (2024)	AAK M-8572, MGU MD-1432
C. bobrinskii	1 ♂, 1 ♀	37.47349	72.58548	puddle in the valley of South Toguzbulok River, 1.5 km east of Lake Turumtaikul	Badakhshan MAR, Tajikistan	Dadykin et al. (2024)	AAK M-8574, ZIN 55621, MGU MD-1433
C. bobrinskii	1 ♂, 4 ♀	40.632559	75.29358	Lake Chatyrkul	Naryn Province, Kyrgyzstan	-	ZIN 50870
C. bobrinskii	4 ♂, 8 ♀	40.997151	76.615361	temporary pool in Muduryum River valley	Naryn Province, Kyrgyzstan	-	ZIN 53837
C. bobrinskii	2 ♂, 2 ♀	46.780759	61.643753	unnamed pool near Aralsk	Kyzylorda Area, Kazakhstan	-	ZIN 30285
C. bobrinskii	many ♂ and ♀	48.687823	71.624296	unnamed pool in Zhanaarka	Ulytau Area, Kazakhstan	-	ZIN 47263
C. altaicusDaday, 1910 sensu Daday, 1910	2 ♂, 1 ♀	51.82163	85.877282	an unnamed waterbody near Sinjukha Mountain	Altai Region, Russia	Daday (1910)	ZIN 9643

Dissections were made using an Olympus SZ-51 optical binocular microscope (Olympus, Japan) and thin steel needles. Intact specimens were photographed using a Leica MZ6 optical binocular microscope with U3CMOS digital camera. Each specimen was photographed with gradual focus shift, with subsequent merging of the image series with Helicon Focus 8 software (Helicon Soft Ltd., Ukraine). Dissected specimens were drawn using a camera lucida mounted on an Olympus CX-41 optical microscope. The micrographs or sketches were inked in Adobe Illustrator 26.0.3 (Adobe Systems Inc., U.S.A.) using a graphic tablet Wacom One CTL-672-N (Wacom, Japan). All measurements were performed in ImageJ software (Schneider et al., 2012).

Scanning electron microscopy specimens were gradually dehydrated in an ethanol-acetone mixture with increasing concentrations (25 %, 35 %, 50 %, 65 %, 75 %, 90 %, 100 %, 100 %) of acetone. The specimens were critical-point dried and coated with gold. Micrographs were taken using a Quattro S scanning electron microscope (Thermo Fischer Scientific, U.S.A.). SEM micrographs were processed and prepared in Adobe Photoshop CC (Adobe Systems Inc., U.S.A.) software. For map creation, QGIS 3.38.0 software (QGIS Development Team, U.S.A.) was applied. For underlying maps, Natural Earth 1:10m vector data (https://www.naturalearthdata.com/downloads/) and ESRI satellite data (https://kmsland.com/esrimap/aerialmap.htm) were used.

Abbreviations and terms

The following abbreviations were used for institutional collections: IM, collection of the Indian Museum, Calcutta, India; AAK, collection of Dr A.A. Kotov, stored in Severtsov Institute of Ecology and Evolution, Moscow, Russia; ZIN, collection of the Zoological Museum of Russian Academy of Sciences, Saint Petersburg, Russia; MGU MD, collection of the Zoological Museum of the Lomonosov Moscow State University, Moscow, Russia.

In diagnoses and descriptions, we followed the terminology of Rogers et al. (2019). Thoracic segments are labeled by Arabic numerals (1-11), abdominal segments by Latin numerals (I-VI). Thoracic appendages are labeled by Latin numerals (PI-PXI). Below, we provide a list of abbreviations used in morphological descriptions for measurements:

AAL, antennal appendage length;

ABL, abdomen length

AL, antenna I length;

ALL, length of antennal appendage anterior lobe;

ANL, antenna II length;

BBL, length of antenna II basomedial branch;

BL, body length;

BSL, length of antennal II proximal antennomere

DAL, distal apophysis length;

DSL, length of antennal II distal antennomere

HL, head length;

HW, head width;

LA, length of anterior portion of the head;

LP, length of posterior portion of the head;

OL, brood pouch length;

OW, brood pouch width;

PAL, proximal apophysis length;

PLL, length of frontal appendage posterior lobe;

TL, thorax length.

The abbreviations used in figures are decoded in the corresponding figure captions.

PCR and sequencing

Genomic DNA was extracted using a modified protocol based on the Wizard Genomic DNA Purification Kit (Promega Corporation, U.S.A.) as follows. A piece of an individual crustacean (two-three thoracic appendages) was dried at room temperature, then placed into the lysis medium (100 µl of Nuclei Lysis Solution, 20 µl of 0.5M EDTA (pH 8.0), and 10 µl of proteinase K (10 mg/ml)), ground against the tube wall, and incubated at 55°C for 18-20 hours. Then, 130 µl of SV Lysis Buffer were added to each specimen and the lysates were processed as in the original protocol except they were washed only twice and the DNA were eluted in a total of 100 µl ultrapure water at 55 °C. The extracted DNA was stored at -20 °C.

The Folmer fragment of the COI mitochondrial gene was amplified using a pair of primers: LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO2198 (5’- TAAACTTCAGGGTGACCAAAAAATCA-3’) (Prosser et al., 2013). An Encyclo Plus PCR kit (Eurogene, Russia) was applied following the manufacturer's protocol and using 2 μl of DNA template. PCR conditions were set as follows: 95 °C for 5 min; 39 cycles: 95 °C for 30 sec, 52 °C for 45 sec, 72 °C for 1 min; 72 °C for 5 min. The CleanMag DNA PCR kit (Eurogene, Russia) standard protocol was applied to purify the PCR products. Bi-directional sequencing of the samples was carried out by Syntol Ltd. (Moscow, Russia). Consensus sequences were assembled using CodonCode Aligner (CodonCode Corporation, U.S.A.). The original sequences were deposited in NCBI GenBank (Sayers et al., 2019), accession numbers PV336111.1-PV336127.1.

Haplotype and phylogenetic analysis

The obtained sequences were aligned by MAFFT (Katoh and Standley, 2013) to assess genetic distances between the samples and all gaps were trimmed with trimAl (Capella-Gutierrez et al., 2009). The TCS haplotype network (Clement et al., 2002) was constructed by PopART (https://popart.maths.otago.ac.nz/) to visualize genetic distances.

Using Unipro UGENE v52.0 (Okonechnikov et al., 2012), we composed a dataset for phylogenetic analysis by examining all the sequences available in the NCBI GenBank for the Folmer region for each species of Chirocephalus (including original data on C. bobrinskii) and Galaziella Naganawa and Orgiljanova, 2000, which is considered a synonym of Chirocephalus, following Rogers (2013; 2024). After aligning the data, we chose representatives of each distinct lineage within the dataset for which uncorrected p-distances reached or exceeded 5 %. Then, the alignment was translated and checked for frameshifts, nonsense mutations and exceptionally high amounts of amino acid substitutions (i.e., for potential mistakes in assembly, NuMTs and other foreign sequences), and dubious sequences were removed. Finally, a sequence of Eubranchipus grubii (GenBank accession number OP596274.1) was added to the dataset as an outgroup. The sequences were aligned by MAFFT (Katoh and Standley, 2013). The phylogenetic tree was built with IQTREE2 (Nguyen et al., 2015) employing 100 standard bootstrap replicates and automatic model selection (Kalyaanamoorthy et al., 2017).

SYSTEMATICS

Class Branchiopoda Latreille, 1817

Order Anostraca Sars, 1867

Family Chirocephalidae Daday, 1910

Genus Chirocephalus Daday, 1910

Chirocephalus bobrinskii ( Alcock, 1898 )

(Figs. 1-10; Fig. S1)

Chirocephalus altaicus - Daday, 1910: 191-195, fig. 22.

Chirocephalus turkestanicus -Daday, 1910: 185-188, fig. 20.

Chirocephalus bobrinskii (Alcock, 1898): 17, fig. 3: 1, 1a (replacement name).

Branchipus bobrinskiiAlcock, 1898: 17, fig. 3: 1, 1a (type locality: Afghanistan, Badakhshan Province, small water body in the vicinity of Lake Chakmaktin). - Daday, 1910: 185-188, 191-195, 212, figs. 20, 22 (altaicus, bobrinskii, turkestanicus). - Kemp, 1911: 220 (bobrinskii). - Vekhoff, 1992: 76-79, figs. 1-4 (altaicus). - Mura, 2001: fig. 2: 3-4 (bobrinskii). - Alekseev and Tsalolikhin, 2010: 439, fig. 239: 1-5 (bobrinskii, turkestanicus). - Dadykin et al., 2024: 16-19, figs. 3, 4C-F (cf. bobrinskii).

Figure 1.
General morphology of Chirocephalus bobrinskii (Alcock, 1898) from near Lake Zorkul (type locality). A-G, female; H-M, male. A, Lateral view; B, head and thorax, dorsal view; C, brood pouch, lateral view; D, E, brood pouch, ventral view: D, mature brood pouch; E, developing brood pouch; F, abdomen, ventral view; G, abdomen, dorsal view; H, adult male, lateral view; I, young male head, lateral view; J, abdomen, ventral view; K, adult gonopods, lateral view; L, adult gonopod basal portion, ventral view; M, adult gonopod retractile portion apex. Abbreviations: 1-13, thoracic segments; I-VI, abdominal segments; asg, anterior shell gland; bmp, gonopod basomedial process; blp, gonopod basolateral process; bp, brood pouch; dls, dorsolateral somatic sensilla; do, dorsal organ; gbp, gonopod basal portion; psg, posterior shell gland; rp, gonopod retractile portion; ts, telson; vls, ventrolateral sensillae. Scale bars: A, B, H = 2 mm; C-G, I, J = 1 mm; K = 500 µm; L-M = 100 µm.

Figure 2.
Head of Chirocephalus bobrinskii (Alcock, 1898) from near Lake Zorkul, Tajikistan (type locality). A, C-I, K, M, N, adult male; B, J, L, O, P, immature male. A, Head, anterior view; B, head, anterior view; C, dorsal organ; D, antenna I distal end, lateral view; E, right antenna II, posterior view; F, right antenna II, medial view; G, apophyses; H, proximal apophysis apex; I, antennal appendage; K-L, antenna II basomedial branch; M-P, variation of antenna II distal antennomere. Abbreviations: AI, antenna I; AII, antenna II; a1, antenna II proximal antennomere; a2, antenna II distal antennomere; aa, antennal appendage; ala, antennal appendage anterior lobe; bb, antenna II basomedial branch; da, distal apophysis; lb, labrum; mla, antennal appendage medial lobe; ne, naupliar eye; pa, proximal apophysis; pla, antennal appendage posterior lobe; st1, antenna I type 1 sensillae; st2, antenna I type 2 sensillae. Scale bars: A = 1 mm; B, E-G, I, M, N = 500 µm; C, J, O, P = 250 µm; D, H, K-L, 100 µm.

Figure 3.
Mouth parts of Chirocephalus bobrinskii (Alcock, 1898) from near Lake Zorkul (type locality), male. A, Labrum, ventral view; B, labrum, lateral view; C, labral outgrowth, ventral view. D, left mandible, medial view; E, left mandible, ventral view; F, left mandible, molar surface; G, right mandible, molar surface; H, left mandible, molar surface outer margin posterior end; I, right mandible, molar surface lateral margin detail; J, maxilla I, ventral view; K, spine at posterodistal angle of maxilla I; L, maxilla II, ventral view; M, maxilla II, sensory element. Abbreviations: a, anterior; d, dorsal; l, labral outgrowth; p, posterior; ps, right mandible posterior spines; pt, left mandible posterior tubercle; v, ventral. Scale bars: A, B, D-G, J, 200 µm; C, L, 100 µm; H-I, 50 µm; K, 25 µm; M, 10 µm.

Figure 4.
Thoracopod morphology of Chirocephalus bobrinskii (Alcock, 1898) from near Lake Zorkul (type locality). A, B, E-L, adult male; C, juvenile male; D, female. A, Thoracic limb I, general view; B-D, limb I endopodite; E, limb I endites 4-6; F, limb I, anterior setae of endite 3; G, limb I, anterior setae of endite 1+2; H, thoracic limb V, general view; I, limb V, endite 2 anterior setae; J, thoracic limb XI, general view; K, limb XI, endites 3-6; L, limb XI, endite 1 anterior setae. Abbreviations: as, anterior setae; e1-6, endites; en, endopodite; ep, epipodite; ex, exopodite; pep1-2, preaepipodites; ps, posterior setae. Scale bars: A, H, J, 500 µm; B-D, 250 µm; E-G, I, K, L, 100 µm.

Figure 5.
Scanning electron microscopy of Chirocephalus bobrinskii (Alcock, 1898) from near Lake Zorkul, Tajikistan (type locality), adult male. A, Antenna I distal part; B-C, right antenna II, medial view; D, right antenna II, posterior view (antennal appendage removed); E, right antennal appendage; F, proximal antennomere anterior surface; G, antenna II sensilla; H, right antenna II distal antennomere; I, adult gonopods, ventral view; J, gonopod basomedial process; K, telson and uropods, dorsal view. Abbreviations: AI, antenna I; AII, antenna II; a1, antenna II proximal antennomere; a2, antenna II distal antennomere; aa, antennal appendage; ala, antennal appendage anterior lobe; bb, antenna II distal antennomere basomedial branch; bmp, gonopod basomedial process; blp, gonopod basolateral process; da, antenna II distal apophysis; pa, antenna II proximal apophysis; mla, antennal appendage medial lobe; pla, antennal appendage posterior lobe; se, sensillae; st1, antenna II type 1sensilla; st2, antenna II type 2 sensilla. Scale bars: A, 100 µm; B-E, I, K, 500 µm; F-H, 250 µm; J, 50 µm.

Figure 6.
Scanning electron microscopy of Chirocephalus bobrinskii (Alcock, 1898) from near Lake Zorkul, Tajikistan (type locality), adult female. A, Head, anterior view; B, antenna I, general view; C, antenna I apex, ventral view; D, right antenna II, anterior view; E, labrum, ventral view; F-G, left mandible, lateral view; H, left mandible, molar processes in posterior portion of ventral margin; I, left mandible molar processes ventral margin, anterior portion. Abbreviations: AI, antenna I; AII, antenna II; do, dorsal organ; lb, labrum; lp, labral process; ms, molar surface; se, antennal sensilla; st1, antenna I type 1 sensilla; st2, antenna I type 2 sensilla. Scale bars: A, 500 µm; B, D, E, 200 µm; C, F, G, 50 µm; H, I, 10 µm.

Figure 7.
Scanning electron microscopy of Chirocephalus bobrinskii (Alcock, 1898). A-H, Female and resting eggs from near Lake Zorkul, Tajikistan (type locality); I-J, resting eggs from valley of South Toguzbulok River, Tajikistan (loc. 9). A, thorax genital segments and abdomen proximal part, lateral view; B, lateral spines and dorsolateral somatic sensillae; C, brood pouch, ventral view; D, distal part of abdomen, telson and cercopods, ventral view; E, abdominal somite VI ventral sensillae row; F, somatic sensilla of abdominal somite VI; G, resting egg, general view; H, resting egg, enlarged part of the surface; I, resting egg, general view; J, resting egg, enlarged part of the surface. Abbreviations: bp, brood pouch; bpo, brood pouch opening; se, sensory element; 11-13, thoracic somites; I-VI, abdominal somites. Scale bars: A-D, 500 µm; E, G, I, 100 µm; F, H, J, 20 µm.

Figure 8.
The TCS haplotype network of specimens morphologically identified as Chirocephalus bobrinskii or C. turkestanicus, based on the Folmer fragment (628 bp) of the COI gene.

Figure 9.
Maximum likelihood phylogram of the genus Chirocephalus based on the Folmer fragment of the COI gene. Branch supports are obtained by bootstrap, the supports higher than 80% are shown in bold. GenBank accession numbers are given in brackets.

Figure 10.
Maps of known records of Chirocephalus bobrinskii (Alcock, 1898) and its junior synonyms. A, General location of the region in Eurasia; B, known range of C. bobrinskii, C. altaicusDaday, 1910 and C. turkestanicus Daday, 1910; C, localities in Tajik and Afghan Pamir. Localities: 1, Lake Chakmaktin, Afghanistan, a type locality of C. bobrinskii according to Alcock (1898). 2-9, Water bodies in Badakhshan MAR, Tajikistan (see Dadykin et al., 2024): 2, 3, two puddles in the valley of Alichur River, 5 km east of Lake Yashilkul; 4, a puddle on the northwest coast of Lake Zorkul, type locality; 5, a lake 3 km east of Lake Zorkul; 6, drying lake 4 km east of Lake Zorkul; 7, a small lake 4 km east of Lake Zorkul; 8, a large lake 5 km east of Lake Zorkul; 9, a puddle in the valley of South Toguzbulok River, 1.5 km east of Lake Turumtaikul. 10, Lake Chatyrkul, Kyrgyzstan. 11, temporary pool in Muduryum River valley, Kyrgyzstan. 12, Unnamed temporary pool near Aralsk, Kazakhstan. 13, unnamed temporary pool in Zhanaarka, Kazakhstan. 14, An unnamed waterbody near Sinjukha Mountain, Altai Region, Russia, a type locality of Chirocephalus altaicus (see Daday, 1910). 15, A small brackish lake near Lake Maraldy, Kazakhstan (see Vekhoff 1992). 16, An unnamed waterbody in Kubergenty mountain pass, Kyrgyzstan (see Daday 1910) (41.233В°N 77.367В°E). White circles indicate populations of C. bobrinskii, white squares C. altaicus Daday, 1910, white triangle C. turkestanicus Daday, 1910 (known only from type locality). Natural Earth 1:10m vector data and ESRI satellite were used as underlying maps.

Type material. Syntypes: males and females. IM 814/10, 1898, S. Alcock coll. (lost). - Neotype: adult male, MGU MD-1434, Tajikistan, Badakhshan Mountainous Autonomous Region, puddle in northwestern coast of Lake Zorkul (37.45477 °N, 73.57547 °E), 2 July 2023, I.A. Dadykin coll. - Paratypes: 7 males and females, MGU MD-1428, Tajikistan, Badakhshan Mountainous Autonomous Region, puddle in northwestern coast of Lake Zorkul (37.45477 °N, 73.57547 °E), 2 July 2023, I.A. Dadykin coll; 7 males and females, MGU MD-1426, Tajikistan, Badakhshan Mountainous Autonomous Region, puddle in the valley of Alichur River, 5 km east to Lake Yashilkul (37.76927 °N 72.99949 °E), 1 July 2023, I.A. Dadykin coll.

Other material. 24 ♂ and 16 ♀ from Badakhshan Mountainous Autonomous Region, Tajikistan (Tab. 1); 1 ♂ and 4 ♀ from Lake Chatyrkul, Naryn Province, Kyrgyzstan (ZIN 50870); 4 ♂ and 8 ♀ from Muduryum River valley, Naryn Province, Kyrgyzstan (ZIN 53837); 2 ♂ and 2 ♀ from unnamed pool near Aralsk, Kyzylorda Region, Kazakhstan (ZIN 30285); multiple ♂ and ♀ from unnamed pool in Zhanaarka, Ulytau Region, Kazakhstan (ZIN 47263). See Tab. 1 for more information on localities and specimens.

Comparative material. Chirocephalus altaicusDaday, 1910, 2 ♂ and 1 juvenile ♀ from an unnamed waterbody near Sinjukha Mountain, Altai Region, Russia (syntypes, ZIN 9643, loc. 14); Chirocephalus chyzeri (Daday, 1890), 2 ♂ and 1 ♀ from Kráľovský Chlmec, Slovakia (ZIN 45175); Chirocephalus spinicaudatus Simon, 1886, 2 ♂ and 5 ♀ from Ivry, France (syntypes, ZIN 15339); Chirocephalus slovacicus Brtek, 1971, 2 ♂ and 2 ♀ from Jesenské, Slovakia (paratypes, ZIN 54297).

Emended diagnosis. Male. Body length 8.5-12.0 mm. Dorsal organ oval, smooth. Male antenna II reaching posteriorly to thoracopod pair III or V. Second antennal appendage trilaminate, serrate, as long as antenna II basal antennomere; antennal appendage medial lamina usually with 3-4 basal digitiform processes, underdeveloped in juveniles. Antenna II proximal antennomere bearing two posteriomedial apophyses, acute basal one and rounded distal one. Antenna II distal antennomere subequal in length to proximal, mature males with antennomere basally arcuate 100 to 120 degrees, juveniles with antenna II distal antennomere slightly arcuate basally. Basal portion in anterior view proximal to arc with basal width 2(( distal width, bearing 2-7 medial spines in mature males, absent in juveniles. Antenna II distal antennomere bearing palmate basomedial branch expanding distally, armed with marginal spines, underdeveloped in juveniles. Labrum with pentagonal proximal portion and short subconical distal portion covered with fine setulae. Thoracic segments lacking spines. Thoracopods I-XI serially homologous but differ in size, each with two serrate praeepipodites, elongate smooth epipodite, exopod and endopod margined with plumose setae, subconic endites 4-6 bearing several anterior setae and a posterior group of plumose setae, and lobiform endites 1+2 and 3, endite 3 with two anterior setae, endite 1+2 with three anterior setae; both endites bearing a posterior row of multiple plumose setae. Thoracopod XI with very short sclerotized praeepipodites and a narrow acute sclerotized epipodite; endite 3 subconic, as large as endites 4-6, bearing 4-5 posterior setae; endite 1+2 similar to that of the thoracopods I-X. Gonopod basal portions close set; gonopod basomedial process elongate, laminate, with inner margin armed by dense short spines. Gonopod basal portion with a small digitiform process located just distal to basomedial process. Gonopod retractile portion nearly smooth, with apex conical and bearing a longitudinal row of 4-5 denticles. Abdomen lacking spines. Abdominal somites I, II, IV and V each with a pair of dorsolateral sensillae; somite II lacking dorsal sensillae; abdominal somite VI with a pair of dorsolateral clusters composed of 7-10 sensillae. Abdominal somites II-V each with a pair of uniform ventrolateral sensillae; somite I lacking sensillae; somite VI with a mediolateral transverse row of 3-5 sensillae. Telson subquadrangular, with a slight posterior emargination; cercopod length 1.5-2 ( abdominal somite VI and telson combined; cercopod margins bearing plumose setae.

Female. Body length 8.5-12.0 mm. Dorsal organ and antenna I as in male. Antenna II as long as the head, unarticulated, subtriangular, with a low basomedial bump. Antenna II anterior surface bearing a subdistal transverse row of sensillae and a basal large sensory field; on the anteromedial surface is a medial transverse row of sensory elements. Thoracic segment 11 with a pair of small dorsolateral spines; segments 7 and 8 with large lateral spines; segments 11-13 with dorsolateral sensillae located at base of the lateral spines. Morphology of thoracic appendages as in male. Brood pouch pyriform elongate, lacking spines. Brood pouch apex reaching or slightly exceeding abdominal segment II posterior margin. Gonopore a terminal slit; a pair of bilobed shell glands located laterally and ventrally. Abdominal somites I, II, III and V each bearing a pair of posteriorly directed lateral spines; abdominal somite IV with two pairs of closely spaced spines. General shape and relative size of the abdominal somites as in male. Telson and cercopods as in male.

Resting egg. Egg diameter 240-340 μm. Mature eggs dark brown, spherical. Egg surface with more or less pronounced wrinkles or fine lacy ornament.

Description. Male. Body length 8.5-12.0 mm. In life having a uniform dark green or dark brown coloration. Head (HL/BL = 0.12-0.15, wide HL/HW = 0.85-0.87), separated by a distinct transverse groove to anterior and posterior part (LA/LP = 1.4-1.8) (Fig. 1 H, I ). Head cuticle smooth, unsclerotized; head frontal margin rounded in dorsal view. Compound eye large (0.3-0.52 mm diameter), spherical, pedunculate (Figs. 1H, I, 2A, B ). Naupliar eye ca. 0.17-0.21 mm diameter, triangular (Fig. 2 A, B ).

Antenna I slightly narrowing distally (AL/HL = 0.80-0.82) (Figs. 2D, 5A, see also Fig. 6 B ). Antenna I surface bearing multiple concentric cuticular rows (Figs. 5A, 6B), absent in proximal and terminal portions. Three subterminal type 1 sensillae, length = 55-60 µm, tapering distally (Figs. 2D, 5A). Ten terminal type 2 sensillae, length = 25-35 µm, unequal in thickness, forming a semicircular row; the central sensillae thickest; each sensilla with a single terminal pore (Fig. 2 D ).

Antenna II varying from long in adults to relatively short in juveniles (ANL/HL = 1.1-1.8), (Figs. 1H, I, 2A B , , S1A-C, E, I). Antenna II proximal antennomere cylindrical, BSL/BSW = 1.8-2.2, slightly curving medially. Proximal antennomere basally with an anteromedial trilaminate antennal appendage. Antenna II proximal antennomere posteriomedial surface bearing two apophyses (Figs. 2E, F, 5B-D, S1A-B (black arrows), C, E, I). Antenna II proximal antennomere anterior surface bearing numerous mechanosensory sensillae medially and distally (Fig. 5 F (white arrow), G). Each sensilla relatively small (10-15 µm diameter), including a seta with folded base, surrounded by a circle of 3-5 tiny cuticular outgrowths (Fig. 5 G ).

Antennal appendage roughly as long as second antennal basal antennomere (AAL/BSL = 0.9-1.2), consisting of three lobes differing in size and structure (Figs. 2F, I, J, 5B (white arrow), C, E). Antennal appendage posterior lobe slightly shorter than anterior lobe (ALL/PLL = 1.1-1.2), subtriangular to subrectangular; posterior lobe lateral margin bearing 8-12 large tubercles with apical denticles directed medially; posterior lobe medial surface covered with small, sparse tubercles (Figs. 2I, J, 5B, C, E). Medial lobe varying in length (MLL/ALL = 0.5-1.1), narrow triangular, usually coiled (Fig. 5B (black arrow), C), with surface covered in tubercles; anterior and posterior margins with short, digitiform outgrowths, crowded basally, becoming more separated distally (Figs. 2I, 5E). Medial lobe anterior margin basally with three to four large, digitiform processes (Fig. 2I) armed with short apical spines (short or absent in juveniles, Fig. 2J). Anterior lobe is the largest, subtriangular, with lateral margin bearing 15-20 large closely spaced apically acute tubercles (Figs. 2I, J, 5C, E). Proximal apophysis located at proximal third of antenna II proximal antennomere (PAL/BSL = 0.55-0.8), conical, straight, subparallel to antennomere axis (Figs. 2E-G, 5B-D); apex acute, armed with short spines and tubercles (Fig. 2G, black arrow, H). Distal apophysis located at distal third of antenna II proximal antennomere, DAL/BSL = 0.25-0.40, cylindrical, oriented perpendicular to antennomere (Figs. 2E-G, 5B-D); apex smooth, broadly rounded (Fig. 5D).

Antenna II distal antennomere not exceeding the length of the proximal antennomere (DSL/BSL = 0.60-0.95), bearing a basomedial palmate branch. Antenna II distal antennomere base slightly to strongly arcuate, directed medially, forming an angle of 100-120° in adult male (Figs. 2A, E, M-P, 5D, H, S1A, B, D, I, J, black arrows); antennomere distal portion width 0.5-0.9x as proximal portion, with distal portion margins subparallel. Antenna II distal antennomere apex rounded in juveniles, truncate or slightly expanded, triangular in cross section in adults (Figs. 2M-P, 5D, H). Distolateral margin with a longitudinal keel, delimited by a deep groove (Fig. 5D). Medial margin proximal third may have several (2-7) small spines at level of basal angle (Figs. 2E, M, N (black arrows), 5D, H). Antenna II distal antennomere basomedial branch lamellar, palmate, explanate (BBL/BSL = 0.28-0.32); distal margin forming 15-20 large spines mostly combined in pairs (Figs. 2K, 5B, D, E, S1D, E (black arrow), F, J, black arrow); spines not pronounced in young individuals (Fig. 2L).

Labrum LL/ANL = 0.60-0.65, flattened dorsoventrally (Fig. 3B); labrum basal portion pentagonal, slightly longer than wide (LL/LW = 1.1-1.3) with lateral margins medially concave (Fig. 3A); proximal angles more prominent than distal. Labral process subconical in ventral view, oval in lateral view, rounded distally, covered with fine setulae (Fig. 3A, black arrow, 3C, see also Fig. 6E, white arrow). Ventral head surface posteriorly to labrum covered by dense setulae.

Mandible large, medially arcuate in ventral view (Fig. 3E); mandible base subcylindrical (Fig. 3D, E). Mandible molar plate posteriorly widely rounded, gradually narrowing anteriorly (Fig. 3D, F, G); right and left molar plates subequal in size. Right and left molar surfaces bearing uniform, minute, closely spaced, lamellar outgrowths, forming 50-60 transverse rows (Fig. 3F-I; see also Fig. 6F-I). Molar outgrowth apices usually worn, except in the most ventral elements. Ventral margin outgrowths digitate, divided into 8-11 elongate, thin lobes (Fig. 6G, H). Marginal element lobes equal in size in posterior half of molar surface (Fig. 5H), but lateralmost lobes gradually increasing in size towards anterior side of mandible, so that anteriormost marginal outgrowths are setiform (Fig. 6G, I). Left and right mandible dorsal margins with asymmetrical armature. Left mandible bearing 8-12 large tubercular teeth along dorsal margin (Fig. 3H, black arrows); posteriormost tooth the largest, located at molar plate posterior end (Fig. 3F, H, see also Fig. 6F, white arrows). Two rows of pits lateral to tubercles, occluded by right mandible outgrowths (Fig. 3H). Left mandible dorsal margin anteriorly with a row of short setae (Fig. 3F). Right mandible dorsal margin armed with ca. 40 transverse rows of teeth, 2-6 in each (Fig. 3G, I); right mandible posterior end with two large spines (Fig. 3G); when worn, the spine rows appear as transverse ridges (Fig. 3G).

Maxilla I subtriangular, spatulate, distal margin armed with a row of 28-30 uniform geniculate setae each bearing several rows of thin setulae (Fig. 3J). The dorsalmost seta of the row 1.3-1.7 ( length of the others (Fig. 3J). Maxilla I ventrodistal angle bearing a short spine (Fig. 3J, black arrow, 3K). Maxilla II subconical (Fig. 3L); apex with two plumose setae; ventrally to the setae is a small, conical sensory element (Fig. 3L (black arrow), M); maxilla II apex covered by dense setulae.

Thorax (TL/BL = 0.40-0.45) lacking spines (Fig. 1H). Each segment with a pair of dorsolateral sensillae, having a similar structure to the sensory elements of antenna II basal antennomere but being larger (ca. 35 µm diameter). Thoracic sensilla pattern as in female (Fig. 1B).

Thoracic limb pairs I-X serially homologous (Fig. 4). Praeepipodite 1 ovoid, lateral margin serrate, ~0.3x in size to praeepipodite 2. Praeepipodite 2 subtriangular with lateral margin serrate (Fig. 4A, H). Epipodite elongate, subcylindrical, width ~0.4 ( length, apex rounded. Exopod subovate, slightly expanded distally, armed with 20-30 long plumose setae of similar structure, decreasing in size towards exopodite base (Fig. 4A, H). Endopod large, subtriangular, with prominent distal angle in anterior limb pairs, becoming more rounded in posterior limb pairs (Fig. 4A, B, H). Lateral margin bearing uniform plumose setae, decreasing in size towards base; medial margin with 5-11 blunt spines, weakly developed in juveniles (Fig. 4B). Thickened plumose setae at spine bases, sometimes abbreviated (Fig. 4B). Exopod and endopod relative size gradually changing across thoracic appendages: in limb I, exopod ~2 ( shorter than endopod (Fig. 4A); in limbs IX and X their length is subequal (Fig. 4H).

Endites 4-6 similar in size, short, conic (Fig. 4A, E, H). Endites 4-6 bearing an anterior cluster of 2-5 short setae and 2-3 posterior long geniculate setae with setulate distal portions; medial margin bearing a longitudinal row of long setulae. Endite 6 bearing 2-5 anterior setae and two posterior setae; endite 5 bearing 2-4 anterior setae and two posterior setae; endite 4 bearing 2-4 anterior setae and three posterior setae (Fig. 4E). Endite 3 short, spatulate (Fig. 4A, H); two anterior setae at proximal third of medial endite margin, the distal seta longer than the proximal; both setae armed with short setulae (Fig. 4F, I). Endite 3 posterior setae numerous (20-30), geniculate, setulose, forming a row intercepting same row of endite 1+2 (Fig. 3A). Endite 1+2 short but very wide, broadly spatulate (Fig. 4A, H). Endite 1+2 bearing three anterior setae: the proximalmost seta located in the middle of the lateral margin, slightly shorter than the posterior setae; two short setae in distal third of the lateral margin, with distal one length 1.5-2 ( the proximal one (Fig. 4G). A row of 90-100 posterior setae similar to that of the endite 2, gradually decreasing in size towards limb base (Fig. 4A, H).

Thoracic limb XI small, with strongly reduced sclerotized praeepipodites (Fig. 4J). Praeepipodite 1 atrophied, a lump; praeepipodite 2 lacrimiform, with apex acute and curved towards epipodite (Fig. 4J). Epipodite narrow, sclerotized, with apex pointed (Fig. 4J). Exopod subequal in size to endopod; endopod subrectangular, widely rounded distally (Fig. 4J). Endites 3-6 similar in size, conical (Fig. 4K); endites 5 and 6 with 4-5 anterior setae and 2 posterior setae; endite 4 with 4-5 anterior setae and three posterior setae; endite 3 bearing 2 anterior setae, one much shorter than the other, and 5-7 posterior setae (Fig. 4K). Endite 1+2 wide, spatulate, bearing three very short anterior setae, located as in other thoracopod pairs (Fig. 4L); a row of 25-40 posterior setae, gradually decreasing in size towards base (Fig. 4J).

Everted gonopod reaching abdominal segment II posterior margin (Figs. 1H, S1K). Gonopod rigid bases close set, directed posteriorly (Figs. 1K, S1K); gonopod rigid basal portion rectangular, bearing proximal subtriangular to subrectangular basomedial process (Figs. 1L, S1H, L); basomedial process sclerotized, directed posteriomedially, with medial margin armed with several rows of short, robust spines (Figs. 1K, L, 5I (white arrow), J, S1H, L). Gonopod rigid bases with a minute, digitiform basolateral process located distally to larger basomedial process (Figs. 1L, 5I, J (white arrows), S1H, K, L, black arrows). Gonopod retractile portion subcylindrical, nearly smooth (Figs. 1K, S1G, K); apex subconical, bearing several subapical minute denticles (Fig. 1M).

Abdomen ABL/BL = 0.25-0.40, smooth, lacking lateral spines. Abdominal somites progressively narrowing posteriorly, segment VI the longest. Location and size of dorsolateral sensillae as in female (see Fig. 1G for female abdomen), except of somite 4 bearing only one pair of sensory elements. Somites II-V each with a pair of small ventrolateral sensillae (ca. 20 µm diameter) (Fig. 1J). Somite VI posterior half dorsally with a pair of lateral sensory fields, 7-10 closely spaced sensillae (20-30 µm diameter) in each; segment ventrally bearing a pair of transverse ventrolateral sensory rows, 4-5 sensillae in each (Figs. 1J, 5K, white arrow).

Telson subrectangular; telson posterior margin with a shallow emargination visible dorsally and ventrally (Figs. 1J, 5K). Cercopod 1.5-2 ( as long as telson and posteriormost abdominal segment combined (Fig. 5K); both margins bear long plumose setae.

Female. Body length 8.5-12.0 mm. Living specimens uniformly dark green or dark brown; mature brood pouch black. Head (HL/BL = 0.15, LA/LP = 1.5-1.6) general morphology and structure as in male, except antennae II (Fig. 6A-D). Antenna II as long as antenna I (AL/ANL = 1.0-1.1), lamellar (Fig. 6A, D). Antenna II slightly flattened dorsoventrally; maximum width in proximal 50 %; lateral margin proximally inflated, forming a sloping bump (Fig. 6A, D). Antenna II apex wide subtriangular, blunt (Fig. 6A, D). Antenna II medial surface bearing a subdistal transverse row of mechanosensory sensillae, similar in size and structure to male antennal sensillae (Fig. 6D, white arrows). Antenna II lateral surface bearing a medial row of sensillae; anterior surface with numerous proximal sensillae (Fig. 6D).

Labrum and mouth parts as in male (Fig. 6E-I).

Thorax TL/BL = 0.42-0.45; segments 12 and 13 bearing brood pouch (Fig. 1A). The first 10 thoracic segments smooth, each bearing a medial pair of dorsal sensillae (Fig. 1B). Each sensilla similar in structure to those of antenna II but larger (ca. 30 µm diameter); each sensilla located in the center of at least two circles of flattened tubercles (Fig. 7A, B, E, F). Thoracic segments 1, 11 and 12 with dorsal sensillae separated by distance equal to 1.5 ( dorsal organ width; segment 2 sensillae closely spaced, separated by a distance equal to compound eye width; sensillae of segments 3-10 separated by a distance equal to ~2 ( compound eye width (Fig. 1B).

Thoracic segments 11-13 each bearing a posteriorly directed lateral spine, each subtended with a dorsal sensilla, ca. 50 µm diameter (Figs. 1A (black arrows), B-C, G (black arrow), 7A, B, white arrows); segment 11 spine subdorsal, 0.25 ( segment 12 spine (Fig. 1G, black arrow); subtending sensilla ca. 30 µm diameter. Segment 12 spine dorsolateral, segment 13 spine lateral (Figs. 1C, 7A, B).

General structure of thoracic appendages as in male but endopodites of anterior limb pairs being wider; also, endopodites of all pairs lacking medial margin spines (Fig. 4D).

Brood pouch reaching or slightly exceeding abdominal segment II posterior margin (OL/BL = 0.21-0.25) (Figs. 1A, C, 7A). Brood pouch smooth, elongate to pyriform (OW/OL = 0.39-0.62), inflated medially, narrowing distally (Figs. 1D, E, 7C). Gonopore terminal (Fig. 1C, 7A (white arrow), C). A pair of bilobed shell glands: smaller anterior portion located at brood pouch base, larger posterior portion in its middle section, at level of abdominal segment II posterior portion (Fig. 1A, C-E).

Abdomen as long as that of male (ABL/BL = 0.30-0.35), segments cylindrical, progressively narrowing posteriorly, segment VI length ~2.5 ( telson length (Fig. 1A, F, G). Abdominal somites I-III, V dorsolaterally each with a pair of spines (Fig. 1A, F, G); segment IV with two pairs of closely spaced spines, the dorsal spine ~0.5 ( the lateral spine (Fig. 1A, G). Abdominal somites I, III-V each with a spine basomedial sensilla (30-50 µm diameter); segment II lacking dorsal sensillae (Fig. 1G). Abdominal somite VI posterior surface with 7-10 scattered lateral sensillae forming a sensory field becoming a transverse row ventrally (Fig. 1G). Abdominal somites II-V ventrally with distolateral sensillae. Abdominal somite I without ventral sensillae; somite II with sensillae ca. 50 µm diameter; somites III-V with sensillae ca. 20 µm diameter (Fig. 1F). Somite VI sensillae clusters ca. 30 µm diameter (Figs. 1F, 7D-F, white arrows).

Telson and cercopod as in male (Fig. 7D).

Resting egg. Resting eggs spherical, 250-340μm in diameter. Developing eggs orange to light brown, mature eggs dark brown. Mature egg surface with variably pronounced wrinkles (Fig. 7G-J), with clusters of minute pores between protuberances (Fig. 7H).

Morphological variability. Individuals display a high level of variability, including morphology of the male antenna II, structure of thoracic limbs, gonopods and brood pouch.

Males vary in the relative size of antenna II. Adult males exhibit the ‘bobrinskii’ morphotype (Fig. 2A) having proportionally larger antennae II; antenna II distal antennomere long, strongly arcuate and often slightly expanded at the apex (Figs. 2M, N, 5D, H, S1D, I, J), with the basomedial branch bearing acute spines (Figs. 2K, S1D-F). Juvenile males represent the ‘turkestanicus’ morphotype and have relatively shorter antennae II with the distal antennomere being only slightly curved, with the apex truncate or even rounded (Fig. 2O, P), and the basomedial branch lacking spines (Fig. 2B, L). Furthermore, the antenna II distal antennomere proximomedial margin will vary in having from zero spines in juveniles to 2-7 spines in adults.

Significant variation is observed in antennal appendage structure as well: the medial lobe may range from 0.5 to 1.1x the length of the anterior lobe (Fig. 2F, I, J). In adults, medial lobe base bears three or four large digitiform lobes, whereas in juveniles the lobes can be short and underdeveloped (Fig. 2J).

A slight variation in seta number occurs on maxilla I (28-30) in both males and females (Fig. 3J).

The endopods of the male anterior thoracopod pairs display strong variation in spine shape and size along medial margin (Fig. 4B, C). Several individuals have deformed endopods, with one or two spines and thickened setae located at the endopod lateral margin, where they are normally absent. Endopod medial margin setae also vary in length, from normally developed (Fig. 4C) to reduced, not reaching the associated spine tips (Fig. 4B).

The number and location of anterior setae on endites 4-6 in both males and females are variable even within a particular pair of appendages: e.g., thoracopod I endite 4 might bear 4-5 anterior setae forming a transverse apical row (Fig. 4A, E), or these setae may form a longitudinal row. In some individuals, the number of anterior setae on endites 4-6 in thoracopods I-X is reduced to 2-3, or several setae are significantly shortened.

The gonopods display significant variation in the shape of the basomedial process, which may be acute (Figs. 1K, 5J) or truncate (Fig. S1K, L) apically. The amount of curve in the basomedial process also varies between populations (Figs. 1K, L, S1G, H, K, L).

The brood pouch significantly differs in size and proportion between young and mature females, being more elongate in immature females and expanded anteriorly in adults (Fig. 1D, E). Mature eggs vary in surface sculpture, from very fine to relatively rough wrinkles (Fig. 7G-J). Size variations are also observed: mature eggs are 250 to 340 μm in diameter and sometimes significantly differ from each other within a particular clutch.

Finally, several males and females have abbreviated cercopods, or only one is normally developed (Fig. 7D) which might be due to predator attacks and subsequent regeneration of these appendages, as was noted for other branchiopods (Hill, 2015).

Genetic variability. Our material shows very low genetic variability, with no differentiation between ‘bobrinskii’ and ‘turkestanicus’ male morphotypes and haplotypes (Fig. 8). Within our material, six haplotypes are present, of which four are presented by single individuals, and the latter two include both ‘turkestanicus’ and ‘bobrinskii’ morphotypes. All haplotypes differ by one or two substitutions only (Fig. 8). The generic assignment of the studied material is confirmed by the phylogenetic analysis (Fig. 9). As far as available genetic data is concerned, the spinicaudatus-group sensu Brtek (1995) is recovered as a strongly supported clade I (97% BS) with C. bobrinskii placed within (Fig. 9).

Type locality. Tajikistan, Badakhshan Mountainous Autonomous Region, puddle in northwestern coast of Lake Zorkul (37.45477°N 73.57547°E).

Distribution and ecology. Chirocephalus bobrinskii (including C. turkestanicus and C. altaicus) is known from the Pamir mountains (Kyrgyzstan, Tajikistan, and Afghanistan), the Russian Altai, and Kazakhstan (Fig. 10A, B). This species occurs in temporary water bodies, including relict brackish lakes (Vekhoff, 1992; this study). Vekhoff (1992) found this species (reported as C. altaicus) in the lowland vicinity of Lake Maraldy in Northeast Kazakhstan, at the end of May. In the Tajik Pamir highlands, C. bobrinskii occurs in the end of June through the beginning of July (Dadykin et al., 2024). Feeding and ecological preferences of the species have not yet been studied.

Remarks. No definitive morphological differences were observed between C. bobrinskii and C. turkestanicus, a species described from Kyrgyzstan based on a single male specimen (Daday, 1910). As Daday (1910) and Alekseev and Tsalolikhin (2010) note, the proximal antennomere of C. turkestanicus lacks the basomedial branch; however, Daday’s original illustrations depict a short outgrowth at anterior side of the second antenna proximal antennomere, near the base of distal antennomere (Daday, 1910: fig. 20a; Alekseev and Tsalolikhin, 2010: fig. 239: 3) which may be an undeveloped basomedial branch, as we found in juvenile C. bobrinskii (Fig. 2G). Rogers et al. (2019) concluded that C. turkestanicus and C. bobrinskii could be distinguished by a shape of antenna II distal antennomere (almost straight in C. turkestanicus and strongly curved in C. bobrinskii). We demonstrate here that this is merely an artefact of age and that the antennomere is actually slightly curved (Fig. 2K, L) and can seem almost straight in medial or outer view (Fig. 2D).

Males of C. bobrinskii are morphologically identical to C. altaicus (ZIN 9643), confirming the synonymy previously suggested by Kemp (1911); however, all our material differs from the C. altaicus material depicted by Vekhoff (1992) which has the distal apophysis elongate and spinose, and by the presence of ventrolateral sensillae (‘brushes of bristles’) only on abdominal segments II-IV (Vekhov, 1992). Our female material of C. bobrinskii and C. altaicus sensu Daday, 1910 has spines on all abdominal segments except VI, whereas in Vekhoff’s (1992) material the spines are absent from segments IV-VI. However, as anostracan sensillae are poorly visible with an optical microscope, those of anterior abdominal segments could have easily been missed by Vekhoff (1992). In other Chirocephalus species, the number of lateral spines was shown to vary even within a particular population (Brancelj and Gorjanc, 1999). General shape of the male second antenna and thoracopods are similar between C. altaicus and C. bobrinskii, as well as the shape of female genital segments, abdomen, and brood pouch (Daday, 1910; Kemp, 1911; Vekhoff, 1992: figs. 1-4). We assume that the presence of spines on the second antenna distal apophysis is insufficient for separating C. altaicus sensu Vekhoff, 1992 from C. bobrinskii.

Chirocephalus bobrinskii belongs to the spinicaudatus species group sensu Brtek (1984; 1995), which is defined by: male antenna II widened basally; antennal appendage trilobate; antenna II basomedial branch spinose; female genital and postgenital segments with lateral spines. Chirocephalus bobrinskii differs from most representatives of Chirocephalus, including a majority of the spinicaudatus group, by having a distal apophysis of male antenna II basal antennomere. Also, C. bobrinskii males differ from those of C. chyzeri, C. slovacicus Brtek, 1971, C. croaticus (Steuer, 1899), C. horribilisSmirnov, 1948, C. robustus Müller, 1966, and C. spinicaudatus Simon, 1886 by the form of the second antenna distal antennomere (Daday, 1910; Vekhoff, 1993). The morphology of maxilla I and maxilla II differ between C. bobrinskii and C. croaticus. Chirocephalus tereki Brtek, 1984 is of very similar appearance to C. bobrinskii but lacks the distal apophysis and the gonopod basolateral process, also the two species show minor differences in the form of the antennal appendage, the female dorsal armature, and the brood pouch. (Brtek, 1984). Within the spinicaudatus group, Chirocephalus povolnyi Brtek, 1967 also possesses two apophyses. Chirocephalus bobrinskii is distinct from C. povolnyi by the absence of spines on the male genital segments and the form of the gonopod retractile portion (Brtek, 1967), although this latter character might be altered due to improper preservation.

At least one more species of Chirocephalus, C. jaxartensisSmirnov, 1948 is known from Central Asia. Males of C. bobrinskii can be distinguished from C. jaxartensis by having a trilobed antennal appendage versus only one lobe in C. jaxartensis and its relatives (Rogers and Soufi, 2013); the form of the second antenna distal antennomere, the shape of the gonopod basomedial process, and the number of spines on the gonopod retractile portion are also suitable for distinguishing the two species (Smirnov, 1948).

Females of C. bobrinskii are more difficult to identify. They can be distinguished from C. chyzeri, C. spinicaudatus, and C. horribilisSmirnov, 1948 by the form of the second antenna (Daday, 1910; Vekhoff, 1993); from C. horribilis by the form of the abdominal spines and by the thoracopod morphology (Smirnov, 1948; Vekhoff, 1993: fig. 4B); from C. croaticus by the morphology of maxillae I and II, the relative size of the brood pouch (large, reaching abdominal somite V in C. croaticus) and the form of the abdominal spines (Brancelj and Gorjanc, 1999); from C. robustus by the presence of a single pair of spines on thoracic segment 11 and the absence of spines on the dorsal brood pouch surface (Brtek 1984). Females of C. tereki are very similar to that of C. bobrinskii, with only few slight differences: in C. tereki the thoracic segment 11 lacks spines, while those of thoracic segment 13 are small; also, abdominal segment IV bears only a single dorsolateral spine in C. tereki. However, these features might be unsuitable for taxonomic purposes due to their possible variability. No obvious morphological differences were observed between females of C. bobrinskii and C. povolnyi (see Brtek, 1967).

The eggs have a surface sculpture very similar to that of C. skorikowi Daday, 1913 (Mura, 2001), but are different from the eggs of C. bobrinskii illustrated by Mura (2001) in having wrinkles (“fine lacy ornament” according to Mura (2001): fig. 2: 3-4).

DISCUSSION

Differentiation, synonymy and phylogenetic position of Chirocephalus bobrinskii

Observed genetic diversity within C. bobrinskii in the Tajik Pamir seems to be very low (Fig. 8, 9). This is not surprising, taking into account that all Pamir localities are relatively close to each other (Fig. 10C). Such low genetic diversity might be due to a ‘bottleneck’ effect (Hewitt, 2004), if the Tajik Pamir populations originated from a small founder population that colonized the high-altitude water bodies of this area (Rogers, 2015).

No reliable morphological or genetic differences were observed between C. bobrinskii, C. turkestanicus, and C. altaicus. We thus treat C. turkestanicus as a junior synonym of C. bobrinskii, and we agree with Kemp (1911) that C. altaicus is also a junior synonym of C. bobrinskii. Males of the “C. turkestanicus” morphotype form a morphological and genetic continuum with the “C. bobrinskii” morphotype, indicating that C. turkestanicus is just a juvenile form of C. bobrinskii. The female of C. turkestanicus was never described, but we found no consistent differences between the females of observed populations. Based on our study, Daday (1910) appears to have described C. turkestanicus based on a juvenile specimen of C. bobrinskii.

Chirocephalus bobrinskii belongs to the spinicaudatus group based on both morphological and genetic evidence, despite knowledge about genetic divergence in Chirocephalus still being very limited (Fig. 9). Males of C. bobrinskii possess the diagnostic characters for the group; however, the classification of Brtek (1995) has been recently challenged (Cottarelli et al., 2017) due to disagreement with the sparse molecular studies conducted so far. Nevertheless, as previously noted (Rogers, 2024), recent molecular analyses attempting to justify the splitting of traditional chirocephalid genera using only a few molecular markers (~ 550 bp of mt16S rDNA and 658 bp of COI gene fragments) and only nine out of more than 70+ species of the family are inadequate for rearranging the Chirocephalidae. Those studies cannot be considered as final and exhaustive, and more research is needed to reconstruct a reliable phylogeny of Chirocephalidae.

We demonstrate that the male second antennal morphology varies with age. Similar intrapopulation size and proportional variability was also reported for Artemia Leach, 1819 (Atashbar et al., 2016; Shadrin and Anufriieva, 2017; Arteaga et al., 2019) depending upon habitat salinity. We strongly advise, therefore, that in addition to molecular tools, culturing methods, or time series wild collections from a single locality (e.g., Rogers, 2003; Rogers et al., 2017), are employed to reduce the possibility that new synonyms are created based on different instars.

Potential value of somatic sensillae for anostracan systematics

The structure, number and location of sensory elements in the Anostraca is often missing from descriptions. These structures include sensory setae of antenna I and II, somatic sensillae and several specific types, such as maxilla II sensillae (Cottarelli et al., 2017).

Antenna I sensory elements are the best known, thus their specific characters are sometimes included in the diagnoses of new species (e.g., Cottarelli et al., 2017). Anostracan antenna I usually bears two types of sensory setae (Tyson and Sullivan, 1979). First, three relatively long type 1 sensillae, lacking pores, which are presumably mechanoreceptors (Tyson and Sullivan, 1979). Second, short terminal type 2 sensillae, each with a single terminal pore, which can be either mechano- or chemoreceptors (Tyson and Sullivan, 1979). The number of the type 2 sensillae varies from 2 to 11 in different representatives of fairy shrimps (Cottarelli et al., 2010, 2017; Rogers and Ferreira, 2007; Rogers and Padhye, 2014). This is also true for C. bobrinskii, which possesses 3 type 1 sensillae and 10 type 2 sensillae (Fig. 6B, C).

However, much less attention has been paid to the somatic sensillae, first described in detail by Tyson and Sullivan (1980) in Artemia. Each of these sensory elements includes a thin central seta with folded base, lacking pores, and a group of cuticular protuberances or ridges surrounding the seta (Tyson and Sullivan, 1980: Fig. 1, 3). Such ‘flower-like’ sensillae are likely to be mechanoreceptors (Tyson and Sullivan, 1980) and might control water currents or relative position of somites, but their role has not been yet studied properly. Similar sensory structures were observed on the antenna II and body surface in a variety of anostracan genera - Artemia, Branchinecta Verrill, 1869, Branchipus Schaeffer, 1766, Tanymastix Simon, 1886, BranchinectellaDaday, 1910, Chirocephalus, Parartemiopsis Rogers, 2005, Streptocephalus Baird, 1852 and Branchinella Sayce, 1903 (Alonso, 1996; Cottarelli et al., 2017: fig. 5D; Shu et al., 2023). In Chirocephalus bobrinskii, these sensillae mostly are located in the positions similar to those in Artemia, a pair of dorsolateral and ventrolateral sensillae per somite (Tyson and Sullivan, 1980; Alonso, 1996). However, the sensory elements of C. bobrinskii vary in size and shape on different somites, and, moreover, the pattern of ventrolateral sensillae is different in males and females (Fig. 1F, J).

Unfortunately, the comparison of C. bobrinskii with its congeners based on these features is still limited due to insufficient knowledge on most Chirocephalus species. Nevertheless, the sensillar pattern of C. bobrinskii significantly differs from that described in Chirocephalus diaphanus Dermarest, 1823 (Alonso, 1996: fig. 16E, F): in the female of C. diaphanus, abdominal somites III-V bear numerous dorsolateral and lateral sensillae (only 1-2 dorsolateral sensillae in C. bobrinskii female). At the same time, both species display similar features contrasting with known sensillar patterns of other anostracan genera (Tyson and Sullivan ,1980; Alonso, 1996; Boudrias and Pires, 2002), namely: 1) presence of dorsolateral sensillae on all abdominal somites except somite II; 2) presence of ventrolateral sensillae on all somites except somite I; 3) sensillae of the somite VI forming a sensory field dorsally and ventrally; 4) all sensillae relatively small, floriform. Somatic sensillae were also observed in Chirocephalus sarpedonisCottarelli, Mura, Ippolito and Marrone, 2017 in positions rather similar to those in C. bobrinskii but their pattern was not described in detail (Cottarelli et al., 2017: fig. 5D). Of course, the abovementioned diagnostic characters are only preliminary, as sensillar patterns need to be verified for other representatives of Chirocephalus.

The somatic sensillae may be a potential source of taxonomically informative features. Although the sensory structures are difficult to discern by optical microscope, they may be suitable for identification of females and juvenile males lacking other essential diagnostic characters. Scanning electron microscopy seems to be a powerful method for revealing these sensillae even in preserved specimens. We hope that this character will be further explored in future taxonomic revisions of anostracans.

ACKNOWLEDGEMENTS

We are grateful to Mario Boboev (Khatlon Science Center, National Academy of Sciences of Tajikistan), Maria O. Ivanova, and Polina A. Volkova (Papanin IBIW, Russian Academy of Sciences), Komyor Boboev, Qobiljon Bobokalonov, Akbar Mamadgoziev, Nuhzor Oshurmamadov, and Khurshed Abdukhalimov for their help with collecting the material. Many thanks to Dr. Victor R. Alekseev and Natalia M. Sukhikh (ZIN, Russian Academy of Sciences) who provided us access to the collection of the Zoological Museum of Russian Academy of Sciences. Also, we would like to thank Ilya I. Turbanov (Papanin IBIW, Russian Academy of Sciences) for providing us with special literature. We are grateful to Sameer Padhye for attempting to find the type material in the Indian Museum. Finally, we express our gratitude to the personnel of the SEM department, Lomonosov MSU (Moscow, Russia) for their amiable treatment and help with processing of the specimens for SEM.

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• Zoobank:
  http://zoobank.org/urn:lsid:zoobank.org:pub:FAEE9D6A-FDB1-484F-B6A1-F6EDE87E7E01
• Consent for publication
  All authors declare that they have reviewed the content of the manuscript and gave their consent to submit the document.
• Funding and grant disclosures
  The study was supported by the Russian Science Foundation (grant № 23-14-00128).
• Study association
  Not applicable.
• Study permits
  The material was collected in compliance with the laws of the Republic of Tajikistan and the Russian Federation. Works with museum collections were coordinated with museum curators, Dr. Victor R. Alekseev (ZIN) and Dr. Elena A. Musatkina (MSU).
• Data availability
  The studied material is deposited in the Zoological Museum of Russian Academy of Sciences, Saint Petersburg, Russia (ZIN 55620, ZIN 55621) and the Zoological Museum of Moscow State University, Moscow, Russia (MGU MD-1426 - MGU MD-1433). All original samples are available in a private collection of Dr A.A. Kotov in the Severtsov IEE of the Russian Academy of Sciences, Moscow, Russia (AAKM). The original sequences are submitted to the NCBI GenBank (access numbers PV336111.1-PV336127.1)

APPENDIX

Figure S1.
Chirocephalus bobrinskii (Alcock, 1898) from various localities in Kazakhstan and Kyrgyzstan, male morphology. A, B, Male from near Aralsk, Kyzylorda Area, Kazakhstan (ZIN 30285); C, male from near Zhanaarka, Ulytau Area, Kazakhstan (ZIN 47263); D, male from Muduryum River valley, Naryn Province, Kyrgyzstan (ZIN 53837); E-H, male from Lake Chatyrkul, Naryn Province, Kyrgyzstan (ZIN 50870); I-L, Chirocephalus altaicus Daday, 1910, male from near Sinjukha Mountain, Altai Region, Russia (type locality, ZIN 9643). A, antenna II in posterior view; B, antenna II, medial view; C, antenna II, posterior view; D, antenna II distal antennomere, anterior view; E, antenna II, medial view; F, antenna II basomedial branch, anterior view; G, gonopods, ventral view; H, gonopod basomedial process, ventral view; I, antenna II, anterior view (antennal appendage not shown); J, antenna II distal antennomere, anterior view; K, gonopods, ventral view; L, gonopod basomedial process, ventral view. Abbreviations: a1, antenna II proximal antennomere; a2, antenna II distal antennomere; aa, antennal appendage; bb, antenna II distal antennomere basomedial branch; bmp, gonopod basomedial process; blp, gonopod basolateral process; da, distal apophysis; gbp, gonopod basal portion; pa, proximal apophysis; rp, gonopod retractile portion. Black arrows indicate diagnostic features of C. bobrinskii. Scale bars = 500 µm.

Edited by

• Editor-in-chief:
  Christopher Tudge

• Editor:
  Lourdes Elmoor Loureiro

Data availability

The studied material is deposited in the Zoological Museum of Russian Academy of Sciences, Saint Petersburg, Russia (ZIN 55620, ZIN 55621) and the Zoological Museum of Moscow State University, Moscow, Russia (MGU MD-1426 - MGU MD-1433). All original samples are available in a private collection of Dr A.A. Kotov in the Severtsov IEE of the Russian Academy of Sciences, Moscow, Russia (AAKM). The original sequences are submitted to the NCBI GenBank (access numbers PV336111.1-PV336127.1)

Publication Dates

• Publication in this collection
  12 June 2026
• Date of issue
  2026

History

• Received
  15 Aug 2025
• Accepted
  09 Nov 2025