Research Article |
Corresponding author: Ivana Karanovic ( ivana@hanyang.ac.kr ) Academic editor: Saskia Brix
© 2017 Ivana Karanovic, Tatiana Ya. Sitnikova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Karanovic I, Sitnikova TY (2017) Morphological and molecular diversity of Lake Baikal candonid ostracods, with description of a new genus. ZooKeys 684: 19-56. https://doi.org/10.3897/zookeys.684.13249
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Uncoupling between molecular and morphological evolution is common in many animal and plant lineages. This is especially frequent among groups living in ancient deep lakes, because these ecosystems promote rapid morphological diversification, and has already been demonstrated for Tanganyika cychlid fishes and Baikal amphipods. Ostracods are also very diverse in these ecosystems, with 107 candonid species described so far from Baikal, majority of them in the genera Candona Baird, 1845 and Pseudocandona Kaufmann, 1900. Here we study their morphological and molecular diversity based on four genes (two nuclear and two mitochondrial), 10 species from the lake, and 28 other species from around the world. The results of our phylogenetic analysis based on a concatenated data set, along with sequence diversity, support only two genetic lineages in the lake and indicate that a majority of the Baikal Candona and Pseudocandona species should be excluded from these genera. We describe a new genus, Mazepovacandona gen. n., to include five Baikal species, all redescribed here. We also amend the diagnosis for the endemic genus Baicalocandona Mazepova, 1972 and redescribe two species. Our study confirms an exceptional morphological diversity of Lake Baikal candonids and shows that both Baikal lineages are closely related to Candona, but only distantly to Pseudocandona.
Crustacea , Deep lakes, molecular phylogeny, taxonomy, CO1, 16S rRNA, 18S rRNA, 28S rRNA
In the past decade the number of Candonidae genera and species has almost doubled, so that now the family contains about 500 Recent species in 39 genera and eight tribes (see
There were several attempts to revise some of the most specious and taxonomically problematic Candonini genera, such as Candona Baird, 1845, Fabaeformiscandona Krstić, 1972, Pseudocandona Kaufmann, 1900, and Typhlocypris Vejdovský, 1882 (see
A majority of 104 Baikal candonids were described in two main publications:
Lake Baikal is a place of exceptional biodiversity. Over 2500 species have been recorded so far, more than half of them endemic to the lake (
In order to recover phylogenetic position of the Lake Baikal candonids within the family we used 10 species from the lake and another 28 from around the word, targeting type species of the genera Candona, Pseudocandona and Fabaeformiscandona, since the majority of the Baikal species currently belong to the former two genera, and all three genera are also currently polyphyletic (
Samples were taken from 11–15 m depths by SCUBA diving from the shore of Lake Baikal. Three bottom types were sampled: rock, mud, and sand. Ostracods were sorted alive on the spot and immediately fixed in 97% ethyl alcohol. Dissection and identification was done with the aid of Zeiss Axiostar-plus light microscope and Leica DM 2500 compound microscope, equipped with N-Plan objectives, respectively. Scanning Electron Microscope (SEM) photographs were taken with a Hitachi S-4700 at Eulji University (Seoul). Photographs of Zenker organ and hemipenis were taken with Olympus C-5050 digital camera mounted on Olympus PX51 compound microscope.
Collected ostracods were identified with the aid of
In the first step of the DNA extraction specimens were kept for 2–3 hours in distilled water. LaboPass Tissue Mini extraction kit (Cosmo Genetech Co., LTD, Korea) was used in all further steps of extraction, following the manufacturer's protocol. Fragments of COI were amplified using universal Folmer primers (
All sequences were visualized using Finch TV version 1.4.0 (http://www.geospiza.com/Products/finchtv.shtml). BLAST (
Mazepovacandona directa (Bronstein, 1947), comb. n.
M. godlewski (Mazepova, 1984), comb.n., M. navitarum (Mazepova, 1976), comb. n., M. orbiculata (Mazepova, 1990), comb. n., M. spicata (Mazepova, 1982), comb. n.
Shell shape variable, but surface generally smooth or poorly ornamented. A1 7- or 6-segmented. Male A2 with t-setae transformed into sensory setae, z-setae transformed into claws. Female A2 G2-claw considerably shorter than G1 or G3. Exopod of A2 consisting of small plate and three setae of which one is long. Male prehensile palps asymmetrical and both with hook-like fingers. L6 with basal seta and with one seta on each endopodal segment, except on last segment, which carries two setae and one claw. L7 with only d1- and dp-setae on basal segment, e- and f-setae missing, g-seta long; terminal segment with short h1-seta and h2- and h3-setae equally long; penultimate segment divided or incompletely divided. UR with both claws and setae present. Zenker organ with variable number of spine whorls, varying from 3+2 to 5+2; anterior part (cap) hemispherical, lattice-like structure well-developed. Hemipenis with small a-lobe not projecting laterally; M-peace terminally rounded (ball-like); ejaculatory process (bursa copulatrix) terminally pointed.
The genus is named after late Dr. Galina Mazepova as an acknowledgment of her outstanding contribution to the study of Lake Baikal ostracod fauna.
Mazepovacandona currently contains five morphologically diverse species. The carapace shape (from triangular to banana shaped) is only one example of this diversity. The number of segments on the antennule and the way male z-setae on the second antenna are developed is also variable, however all females have G2-claw on the second antenna shorter than the rest of the claws. The number of setae on the second segment of the Md-palp is also variable and it can be either three or four. Prehensile palps are dissimilar among species, although all have clearly pronounced hooked-like fingers on both left and right palp. The basal seta (d1) on the walking leg is shorter in all five species than in two Baicalocandona species redescribed here. The length of this seta relative to the d2-seta (always absent in Candonidae) is an important taxonomic character in some Cyprididae, such as Cyprinotinae (see the key in
Despite the morphological diversity of Mazepovacandona, this genus seems to be most closely related to Candona and Fabaeformiscandona. For example, prehensile palps of M. directa (elongated) are very similar to candida-group of Candona, while female genital lobe bears similarity to the neglecta-group. There is also similarity with Fabaeformiscandona, especially because several of its species have rounded distal part of the M-peace. The breuli-group of the latter genus is particularly similar to Mazepovacandona in sense that the M-peace is not so strongly sclerified and that most species have an UR with a long posterior seta. However, most of the species currently belonging to this group have a completely fused penultimate segment of the cleaning leg.
Candona
directa
sp. n. –
Candona
directa
Bronstein –
Two males and one female dissected and mounted on glass slides (shell of one male and one female on one SEM stub), 25 undissected specimens in 95% alcohol, 1 specimen used for the DNA extraction, all collected from 12–15 m depth by SCUBA diving off Listvyanka, 51°51'51.3"N, 104°50'37.8"E, 12 September 2015, collectors: Igor Khanaev and Ivan Nebesnykh.
Almost no sexual dimorphism in shell shape in lateral view (Fig.
A1 7-segmented with posterior setae transformed into claws (Fig.
Candona
godlewski
sp. n. –
Candona
godlewski
Mazepova –
One females dissected and mounted on glass slides (shell on the SEM stub), 2 undissected specimens in 95% alcohol, 2 specimens used for the DNA extraction), all collected from 12–15 m depth by SCUBA diving off Listvyanka, 51°51'51.3"N, 104°50'37.8"E, 12 September 2015, collectors: Igor Khanaev and Ivan Nebesnykh.
Both LV and RV banana shaped (Fig.
A1 7-segmented, with segments 3 and 4 partly fused (Fig.
Females not collected.
Baicalocandona
navitarum
sp. n. –
Baicalocandona
navitarum
Mazepova –
One male soft body used for DNA extraction and after that dissected and mounted on a glass slide (shell of one SEM stub), collected from 12–15 m depth by SCUBA diving off Listvyanka, 51°51'51.3"N, 104°50'37.8"E, 12 September 2015, collectors: Igor Khanaev and Ivan Nebesnykh.
Valves asymmetrical: LV subtriangular with pointed dorsal margin, RV with rounded dorsal margin (Fig.
A1 7-segmented. Male A2 with subdivided penultimate segment and t2- and t3-setae transformed into sexual bristles; z2-setae transformed into claw, z1- and z3-setae untransformed; G1- and G3-claws reduced and short, G2-claw long (Fig.
Females not collected.
Candona
orbiculata
sp. n. –
One male soft body used for DNA extraction and after that dissected and mounted on one glass slide (shell on SEM stub), three juveniles kept in 95% alcohol, all collected from 12–15 m depth by SCUBA diving off Listvyanka, 51°51'51.3"N, 104°50'37.8"E, 12 September 2015, collectors: Igor Khanaev and Ivan Nebesnykh.
Valves reniform in lateral view, with almost evenly rounded dorsal margin (Fig.
A1 lost during DNA extraction. Male A2 with subdivided penultimate segment and t2- and t3-setae transformed into sexual bristles; z1-seta transformed into claw, z2-setae not observed, z3-seta untransformed; G1- and G3-claws reduced and short, G2-claw long (Fig.
Candona
spicata
sp. n. –
Candona
spicata
Mazepova –
One male soft body used for DNA extraction and after that dissected and mounted on one glass slide (shell of one SEM stub), one juvenile kept in 95% alcohol, all collected from 12–15 m depth by SCUBA diving off Listvyanka, 51°51'51.3"N, 104°50'37.8"E, 12 September 2015, collectors: Igor Khanaev and Ivan Nebesnykh.
Valves elongated in lateral view, with almost straight dorsal margin (Fig.
A1 7-segmented, some posterior setae transformed into claws (Fig.
Baicalocandona bivia Mazepova, 1976
Shell shape (always) trapezoidal, surface ornamented in most species, at least in some parts. A1 7, 6 or 5-segmented. Male A2 with t-setae transformed into sensory setae, z-setae transformed into claws. Female A2 with G2-claw as long as G1 or G3. Exopod of A2 consisting of small plate and three setae of which one long. Male prehensile palps asymmetrical and both with hook-like fingers, but right palp with shorter, stockier and considerably less hook-like finger. L6 with basal seta and with one seta on each endopodal segment, except last, which carries two setae and one claw. L7 with only d1- and dp-seta on basal segment, e- and f-setae missing, g-seta long; terminal segment with short h1-seta and h2- and h3-setae equally long; penultimate segment fused without any notable subdivision. UR with both claws and setae present. Zenker organ with 4+2 whorls of spines. The anterior part (cap) more hemispherical and margin not sclerotized, lattice-like structure not well-developed; cap also with long radiating spine-like projections. Hemipenis with relatively large a-lobe not projecting laterally. M-peace terminally foot-like; ejaculatory process (bursa copulatrix) not terminally pointed, and with broad, rounded, finger-like extension; this process also with lateral thorn-like ornamented part.
Baicalocandona at the moment includes 11 species and 11 subspecies. According to the diagnosis (
Candona
rupestris
dissona
subsp. n. –
Soft parts of one male and one female used for DNA extraction, after that each dissected and mounted onto one glass slides, their shells kept on one SEM stub each, 40 juveniles kept in 95% alcohol, all collected from 12–15 m depth by SCUBA diving off Listvyanka, 51°51'51.3"N, 104°50'37.8"E, 12 September 2015, collectors: Igor Khanaev and Ivan Nebesnykh.
Almost no sexual dimorphism in shell shape in lateral view (Fig.
A1 7-segmented (Fig.
Soft parts of one male used for DNA extraction, after that dissected and mounted onto one glass slide, shell broken during dissection, collected from 12–15 m depth by SCUBA diving off Listvyanka, 51°51'51.3"N, 104°50'37.8"E, 12 September 2015, collectors: Igor Khanaev and Ivan Nebesnykh.
A1 6-segmented. Male A2 with subdivided penultimate segment and t2- and t3-setae transformed into sexual bristles; both z1- z2-setae transformed onto claws; G1- and G3-claws reduced and short, G2-claw long. Md-palp with 4+2 setae on the inner side, gamma seta not plumose. Prehensile palps stocky and right one with a very strong finger but not hook-like. Hemipenis (Fig.
BLAST analyses of the GenBank database revealed that the obtained sequences were ostracod in origin and not contaminants. No stop codons were detected in the COI sequences. The COI alignment was 672 base pairs long, and included four species each with one sequence. The concatenated dataset was 3302 base pairs long, and it included 50 sequences belonging to 39 species. Of the individual alignments, 18S dataset was the longest (1042 positions) and also included 50 terminals. The alignment of 16S was the shortest (554 base pairs), and had only 21 species. After the exclusion of ambiguous blocks, 28S alignments varied from 660 base pairs (em fragment) to 455 base pairs (df fragment). The vx primer pair was the most successful in amplifying the region, while df fragment was very difficult to amplify and only 34 sequences were analyzed. The amplification by em primer pair was relatively successful, but this was the most difficult dataset to aligned due to the long expansion segments present in several species. Although initially this alignment was very long (1521 base pairs), after the Gblock analysis (
GTR (
The results of p-distance analysis are shown in Fig.
After two million generation runs in MrBayes, the final standard deviation of split frequencies fell below 0.01 (it was around 0.003) and the potential scale reduction factor was ~1.0 for all parameters, suggesting that convergence had been reached. All resulting consensus trees were rooted with the outgroup, Physocypria sp. Fig.
50% majority rule consensus tree of the family Candonidae constructed from the concatenated dataset of two nuclear (18S & 28S) and one mitochondrial (16S) markers. Numbers on the branches represent Bayesian posterior probabilities. Underlined taxa represent type species. Grey shaded taxa are Lake Baikal species. Tree rooted with Physocypria sp. Tribes are labeled with letters: ACandoniniBCryptocandoniniCCandonopsiniDTrapezicandoniniEHumphreyscandonini.
The larger clade on the tree was composed of two tribes. All except Cryptocandona smithi Karanovic & Lee, 2012 belong to the largest Candonidae tribe, Candonini. Candonini can be broadly divided into three clades, all with maximum posterior probabilities. Ten Lake Baikal candonids did not form a monophyletic clade, but clustered with some non-Baikal species, in particular Fabaeformiscandona kushiroensis, Candona candida, C. bimucronata, and C. neglecta. Fabaeformiscandona kushiroensis is nested within the Mazepovacandona clade. The clade composed of the second Baikal lineage and three Candona species received a very low support (below 0.5 posterior probability). A clade composed of nine species belonging to Candona, Pseudocandona, and Typhlocypris was sister to the previous, mostly composed of Baikal candonids, but this association did not have high posterior probability (0.7). The last group on the tree, consisting of Earicandona and Fabaeformiscandona, was strongly supported and was sister to the previous two clades.
When defining Baikal genera, we were mostly lead by the results of the molecular phylogeny analysis, which indicated that the 10 Baikal species belong to only two lineages. However, the morphological diversity of Lake Baikal candonids is extraordinary, especially when compared with the candonid fauna from other parts of the world. In fact, when compared with the Holarctic candonid genera, each Mazepovacandona species redescribed in this paper has enough apomorphic characters (from the shell shape to the number of whorls on the Zenker organ) to be described in a separate genus. In addition,
Based on our phylogenetic tree, none of the Baikal species included in this study could be assigned to either Candona or Pseudocandona, as demonstrated by the position of the type species of these two genera (underlined species on the tree). Nevertheless, they are morphologically and genetically more closely related to Candona than to any other Candonidae genera included in this analysis. Candona is a polyphyletic taxon, which is illustrated by the fact that most (if not all) of the Candona species endemic to Baikal Lake should be excluded from it, and by the position of C. quasiakaina Karanovic & Lee, 2012 nested within the true Pseudocandona/Typhlocypris clade on the tree. Fabaeformiscandona is also a polyphyletic genus, which was already speculated many times (
Molecular diversity of gene markers commonly used for resolving higher phylogenetic relationships (18S and 28S) is relatively small between Baikal candonids and their closest relatives, in comparison to other ostracod lineages. For example, in the family Cyprididae, distances between 18S sequences vary from 2% (within genus) to 11% (between genera) (Kong et al. 2014); while in Polycopidae the same marker has approximately 3% intragenic and 10% intergeneric variability (
The study is supported by the National Research Foundation of Korea (grant no: 2016R1D1A1B01009806). We would like to thank Igor Khanaev and Ivan Nebesnykh from the Limnological Institute, Siberian Branch, Russian Academy of Sciences for collecting the samples. The work was partly support by Russian Government funded project No. 0345-2016-0009.
Locality data and BenBank Accession Numbers
Data type: molecular data
Explanation note: Species in bold are our sequences, regular font species were downloaded from the GenBank.
p-distances between 18S rRNA sequences
Data type: statistical data
Explanation note: data were used for the Figure
p-distances between 28S rRNA sequences, df region
Data type: statistical data
Explanation note: data were used for the Figure
p-distances between 28S rRNA sequences, em region
Data type: statistical data
Explanation note: data were used for the Figure
p-distances between 28S rRNA sequences, vx region
Data type: statistical data
Explanation note: data were used for the Figure
p-distances between 16S rRNA sequences
Data type: statistical data
Explanation note: data were used for the Figure
p-distances between COI sequences
Data type: statistical data
Explanation note: data were used for the Figure