Liu et al. Zool. Res. 2021, 42(3): 287−293 https://doi.org/10.24272/j.issn.2095-8137.2020.318 Letter to the editor Open Access Does the Dzungarian racerunner (Eremias dzungarica Orlova, Poyarkov, Chirikova, Nazarov, Munkhbaatar, Munkhbayar & Terbish, 2017) occur in China? Species delimitation and identification with DNA barcoding and morphometric analyses The Eremias multiocellata-przewalskii species complex is a dzungarica, which were primarily associated with sexual viviparous group in the genus Eremias, and a well-known dimorphism and a broader range of values for various traits. representative of taxonomically complicated taxa. Within this The rapid development of DNA barcoding (Hebert et al., complex, a new species – E. dzungarica (Orlova et al., 2017) 2003) has facilitated the successful application of a – has been described recently from western Mongolia and standardized short mitochondrial gene fragment, COI, to most eastern Kazakhstan, with an apparent distribution gap in species level identifications (e.g., excluding plants), species northwestern China. In this study, we used an integrative discovery and global biodiversity assessment (DeSalle & taxonomic framework to address whether E. dzungarica Goldstein, 2019; Yang et al., 2020). DNA barcoding is indeed occurs in China. Thirty specimens previously classified particularly helpful for phylogenetic and taxonomic inference in as E. multiocellata were collected in eastern Kazakhstan and species groups that have considerable morphological the adjacent Altay region in China. The cytochrome c oxidase conservatism or ambiguity (e.g., Hofmann et al., 2019; Oba et I (COI) barcodes were sequenced and compiled with those al., 2015; Xu et al., 2020; Zhang et al., 2018). from Orlova et al. (2017) and analyzed with the standard and Traditional taxonomy is mainly based on morphological diverse barcoding techniques. We detected an absence of a characters, and hence can easily lead to misidentification as a barcoding gap in this complex, which indicates potential result of phenotypic plasticity, cryptic species or different cryptic species in Eremias sp. 3 with high intraspecific morphologies at different life history stages (Bickford et al., diversity and multiple recently evolved species in Clade A. 2007; Lee, 2004; Rock et al., 2008). Taxonomy relying on Both BIN and GMYC suggested an unrealistically large DNA barcodes alone is also unrealistic, as mitochondrial number of species (23 and 26, respectively), while ABGD, genes have many inherent biases and limitations in species mPTP and BPP indicated a more conservative number of delimitation, e.g., those associated with maternal inheritance, species (10, 12, and 15, respectively), largely concordant with reduced population size, inconsistent mutation rate, or the previously defined species-level lineages according to evolutionary processes such as purifying selection (Blair & phylogenetic trees. Based on molecular phylogeny and Bryson, 2017; Pino-Bodas et al., 2013; Rubinoff et al., 2006). morphological examination, all 30 individuals collected in this With the advance of barcoding techniques, however, the study were reliably identified as E. dzungarica – a distinct evidence inferred from DNA barcoding can guide further species – confirming the occurrence of this species in the targeted morphological analysis, and the use of multiple lines Altay region, Xinjiang, China. Potentially owing to the larger sample size in this study, our morphological analyses revealed of evidence, such as nuclear loci, geographical and ecological many inconsistencies with the original descriptions of E. data to make more robust inferences about the species boundaries under the rubric of integrative taxonomy (Damm et This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// Received: 01 November 2020; Accepted: 06 April 2021; Online: 07 creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted April 2021 non-commercial use, distribution, and reproduction in any medium, Foundation items: This work was supported by the Strategic Priority provided the original work is properly cited. Research Program of the Chinese Academy of Sciences Copyright ©2021 Editorial Office of Zoological Research, Kunming (XDA20050201) and National Natural Science Foundation of China Institute of Zoology, Chinese Academy of Sciences (32070433 to X.G.G. and 32000288 to J.L.L.) al., 2010; Dayrat, 2005; Miller, 2007; Padial et al., 2010; Will et and propose putative species in the E. multiocellata- al., 2005). przewalskii species complex based on mitochondrial lineages, The Eremias multiocellata-przewalskii species complex incomplete morphological identification characters (e.g., no comprises a natural group of viviparous species in the genus voucher specimens from certain lineages) and geographic Eremias (Guo et al., 2011; Orlova et al., 2017). The taxonomy distributions. However, they did not utilize more rigorous of this species complex has been historically confusing due to barcoding techniques to deeply explore the distribution of the vast phenotypic variation within and among species, as genetic distance and to test species boundaries in this species well as the conservation of morphological characters in closely complex. To determine whether E. dzungarica occurs in related species (Eremchenko et al., 1992; Eremchenko & China, we sequenced the DNA barcoding COI fragments and Panfilov, 1999). So far, as many as 18 species/subspecies performed morphological measurement of the 30 purported E. have been proposed across its wide distribution range multiocellata individuals collected from seven locations in covering Kyrgyzstan, eastern Kazakhstan, northern China, eastern Kazakhstan and the adjacent western Altay region, Mongolia, and southern Tuva Republic of Russia (Orlova et Xinjiang, China (Figure 1A; Supplementary Table S1). al., 2017; and references therein). Among these species is the Subsequently, sequences of the species complex from Orlova newly delimited Dzungarian racerunner, Eremias dzungarica et al. (2017) were compiled and analyzed with diverse (Orlova et al., 2017). In addition to the apparent molecular and commonly used barcoding methods to explore the intra- and morphological deviations from congeners as described in interspecific genetic distance patterns and reassess the Orlova et al. (2017), this species is characterized by a habitat species status of the species-level lineages proposed by preference for rocky hills and gravel ravines ( “rocky form" Orlova et al. (2017). Finally, we explicitly identified the coined in Orlova et al. (2017)) in western Mongolia at high taxonomic status of the “multi-ocellated racerunners” collected elevations (2 400−2 600 m above sea level (a.s.l.)). However, from Kazakhstan and China with molecular and morphological it can also penetrate into low-altitude sandy dune areas in data. Detailed methods are available in the Supplementary eastern Kazakhstan (400−1 000 m a.s.l.). As such, it remains Materials and Methods. unclear as to whether E. dzungarica occurs in the vast Sequences from Orlova et al. (2017) had different lengths; territories of the northern Junggar Depression in Xinjiang, most of the sequences (82.3%) were 651 bp, however some China, between western Mongolia and eastern Kazakhstan. were 617−619 bp with missing data located at both ends of To date, four occurrences of so-called E. multiocellata (the the sequences. To accommodate the majority of these multio-cellated racerunner) have been recorded from only two sequences, a dataset of 651 sites was generated. A total of 81 regions in the northern Junggar Depression: one in the haplotypes were determined, including four for the outgroups. Tacheng region and three in the Altay region reported in Zhao 235 sites were variable, and 190 were phylogeny-informative. (1999) and Tao et al. (2018), respectively. As suggested by The general trends of Kimura 2-parameter genetic distance Orlova et al. (2017), E. dzungarica may have been considered indicated an increment from lower to higher taxonomic levels as E. multiocellata in China, hence it is possible that these (Supplementary Figure S1). However, the genetic distances at reported populations are in fact E. dzungarica, despite the lack the species and genus level overlapped at a low frequency of morphological and molecular data. (Supplementary Figure S1), indicating no apparent barcoding Orlova et al. (2017) for the first time utilized the DNA gap between intra- and interspecific distances. Intraspecific barcoding sequences (COI) to infer phylogenetic relationships distances ranged from 0 to 6.18% (mean±standard deviation A E E E E 26 RUSSIA 47 N 56 57 58 62 MONGOLIA 41 KAZAKHSTAN 55 61 53 52 59 40 60 54 46 25 24 23 50 22 48 45 44 38 Xinjiang 51 49 43 42 39 37 27 29 8 9 28 4 5 3 6 7 2 11 13 KGZ 1 10 Inner Mongolia 12 21 36 CHINA Gansu 32 35 N 14 33 19 20 34 18 31 15 Qinghai 16 17 30 Key: E. szczerbaki E. stummeri E. yarkandensis E. cf. buechneri Eremias sp. 1 Eremias sp. 2 E. cf. multiocellata Eremias sp. 3 E. przewalskii E. cf. przewalskii E. cf. reticulata E. multiocellata E. dzungarica (E Kazakhstan; W Mongolia; 52−55) E. dzungarica (China: Altay, Junggar Depression; E Kazakhstan; 56−62) KGZ: Kyrgyzstan 288 www.zoores.ac.cn B Hap 13 1 2 3 4 5 Hap 14 Hap 8 Hap 11 67/63/0.88 Hap 5 Hap 9 China: Altay, Junggar Hap 2 68/56/0.88 Hap 1 Depression; E. dzungarica Hap 6 E Kazakhstan Hap 7 (56−62) 98/96/1.0 Hap 10 Hap 15 Hap 4 Hap 12 Hap 3 57/−/− Hap 29 Hap 30 E Kazakhstan; W Hap 33 Mongolia; Clade A −/−/0.57−Hap 31 Hap 32 (52−55) Hap 28 E. cf. buechneri China: Qarqan 64/−/0.52 Hap 48 E. yarkandensis SE Kyrgyzstan 1 BIN −/−/− Hap 16 E. przewalskii Hap 17 91/78/1.0 Hap 20 China: Alashan desert; Hap 18 C and N-W Mongolia; 2 GMYC Hap 19 Russia: Tuva Republic Hap 24 E. cf. przewalskii 97/−/0.91 Hap 25 S Mongolia 3 ABGD 90/75/0.98 Hap 26 Eremias sp. 2 Hap 27 China: Qinghai 95/89/1.0 Hap 23 99/92/0.99 Hap 21 E.