Hybridization in Selected Species and Genera of Diaptomid Copepods in China

: To better understand the fauna of freshwater calanoid copepods of China, including the occurrence of intra- and intergeneric hybridization, we studied ﬁve species, distributed across the whole of China or in South China. We sequenced a mitochondrial (COI) and the nuclear ribosome 18S operon (ITS) to reconstruct the phylogenetic trees by using a Bayesian and maximum likelihood (ML) approach with 161 individuals. The phylogeny tree revealed ﬁve clades and two geographically separated subclades in both S. ferus and P. tunguidus . We found, for the ﬁrst time, that the hybrid specimens occurred in Diaptomidae, but low hybridization suggested effective barriers to hybridization and introgression. One hypothesis, that hybridization is recent and was initiated by invasions via canals built between the Yangtze and Pearl rivers c. 2000 years ago, is not supported by K2P genetic distances of the order of 20%. Furthermore, COI analysis of different populations of S. ferus and P. tunguidus revealed two geographical clades in each species, with genetic distances commensurate with cryptic speciation. Both clades occupy subranges maintained without visible barriers to mixis.


Introduction
Formation of new species or merging of species by hybridization are interesting phenomena [1,2]. In freshwater Zooplankton, natural hybridization is common in the Cladocera [3][4][5], while Parent et al. (2012) found hybrids between Calanus finmarchicus and C. glacialis (Copepoda, Calanoida) in the Arctic and Northwest Atlantic [1]. In freshwater, the family Diaptomidae is the dominant taxon of the calanoida. Its taxonomy, both at the genus and the species level, relies heavily on the morphology of the fifth thoracic limb (P5) in males [6,7]. Historically, hybrid status of specimens was inferred from morphological analysis and ecological context, for example, in specimens morphologically intermediate and found in the zone of contact between the parental species [8]. However, for zooplankton species, it may be hard to find intermediacy in tiny characters. In fact, it should be shown that putative hybrids have genetic information of the two parental species. Hybridization has recently been suggested, using appropriate molecular techniques, between subspecies of Eodiaptomus endemic to the Malili lakes of Sulawesi [9].
The internal transcribed spacers (ITS) of the 18S nuclear ribosomal DNA have been considered a useful molecular marker for hybrids [10,11]. Mitochondrial DNA is a circular molecule, with parental inheritance, a rapid evolutionary rate, and a large number of copies. It has been widely used for analyses of metazoan phylogenetic relationships at various taxonomic levels [12][13][14][15].
The freshwater Diaptomidae of China comprise 16 genera, with about 50 species [16]. Diaptomidae tend to have restricted ranges that may encompass several river basins, a single river basin, or part of a river basin. One of the species discussed here, Sinodiaptomus cavernicolax, is even restricted to a short underground river in Longyan, a cave which is part of the Star Lakes complex of Zhaoqing city, Guangdong province. Sinodiaptomus ferus is found in the Star lakes and in some reservoirs in the same general area. It also penetrates Longyan cave and meets S. cavernicolax there [17]. Females of the two can only be distinguished by body size. Sinodiaptomus sarsi, a third Chinese species of the genus, is widespread over China but with limited penetration in the west. Phyllodiaptomus tunguidus is widely distributed in the Yangtze but especially the Pearl River basin, including Star Lakes, where it is syntopic with the two aforementioned Sinodiaptomus species but has never been recorded in the cave. Neodiaptomus schmackeri also often coexists with P. tunguidus in South China. Coexistence of species and genera makes it theoretically possible for hybridization to occur. To evaluate this expectation, we used a combination of mitochondrial and nuclear DNA markers.

Materials and Methods
Thirteen sites were sampled from 6 provinces in China between 2011 and 2016 ( Figure 1 and Table 1). Special attention was paid to Star Lakes (23 • 6 N 112 • 23 , Zhaoqing City, China), a complex composed of five shallow lakes (Bohai, Zhongxin, Qinglian, Fairy and Li). Longyan cave contains a shallow lake, a few hundreds of meters long and ending at the entrance of the cave. The distance between lake Li and the cave is not more than 10 m [17]. Five samplings in Star Lakes were carried out: in November 2011, July and November 2012, and again in October and November 2013, and May 2015. Zooplankton was collected with a plankton net of 120 µm mesh size, towed from the bottom to the surface. All samples were preserved in ethanol 95% at 4 • C.
The authenticity of COI and ITS sequences were verified by a BLAST search in Gen-Bank. The sequences were edited in Mega 7.0 (Institute for Genomics and Evolutionary Medicine, Temple University, USA) and Finch TV 1.5.0 (Geospiza Research Team, USA). The total length of the sequence segments after alignment were 555 bp for COI and 652 bp for ITS. We used Acanthodiaptomus pacificus as the outgroup species.
The best-fitting model selected by MRMODELTEST 2.3 for the ITS dataset was of GTR+G with a relative AIC weight of 0.5110 and gamma distribution shape parameter 0.4060. The best-fitting model for the COI dataset was GTR+I+G with a relative AIC weight of 0.7905 and gamma distribution shape parameter 0.7180.
Bayesian inference and ML for both genes revealed the same five clades: N. schmackeri (A), P. tunguidus (B), S. sarsi (C), S. ferus (D), S. cavernicolax (E) (Figures 2 and 3). However, while in the COI tree, all specimens were recovered consistent with their morphological taxonomy (Figure 2A,B); in the ITS tree, some specimens end up in a "wrong" cluster ( Figure 3A,B). The COI gene sequence always recovers the mother; if the ITS gene finds another species, the specimen in question is a hybrid. In Clade A of the ITS tree, No.9 S. ferus, for example, is a hybrid with N. schmackeri. Clade B contains one hybrid of S. × cavernicolax. Clade D contains two well-supported sublineages from Zhuyin reservoir, Zhuhai city (D1) and Star Lakes, Zhaoqing city (D2). In this clade, there are four putative hybrid specimens of S. × cavernicolax and one P. × tunguidus. Clade E contains three hybrids of S. × ferus. The COI tree reveals the same five well-supported main clades as the ITS tree. Five of ten hybrids form a clade of their own, with strong bootstrap support. In the COI tree, Clade B and D both contain two well-supported sublineages from the Guangdong province population (B1) and Guangxi province population (B2), Zhuyin reservoir, Zhuhai city (D1) and Star Lakes, Zhaoqing city (D2), both in Guangdong province.

COI Divergence
The K2P pairwise distances derived from COI varied between 11% and 32.6%. The highest distance was between clade C (S. sarsi) and clade D1 (S. ferus); the lowest was between clade B1 (P. tunguidus, Guangdong population) and B2 (Guangxi population). The second lowest genetic distance, 15.1%, occurred between clade D2 (S. ferus) and clade E (S. cavernicolax). The distance between clades D1 and D2 reached 17.8% even though they were same morphospecies, higher than between clade E and D2, and also higher than the average intra-specific variation in Crustacea [27]. The average genetic distance between genera was 21.1%; within genera it was 21.8% (Table 2).

Discussion
Hybrids in freshwater microcrustaceans are hard to identify by morphology, despite a high genetic divergence, which surely contributes to their apparent rarity. However, our results show that N. schmackeri, P. tunguidus, S. ferus and S. cavernicolax do hybridize, but hybridization rates are low, of an order of 5.4% between species and of 2.3% between genera. These results suggest the operation of rather effective pre-or post-mating barriers, and introgression is unlikely. At this stage, it is not possible to ascertain whether the hybrids are fertile or not.
S. cavernicolax is not, as its describers claimed (Shen and Tai, 1965), a true stygobiont, because it is not depigmented and has eyes. However, it has never been found outside Longyan cave. Hybrids between the congeners S. ferus and S. cavernicolax are therefore by necessity limited to the cave where the two coexist. S. ferus has a fairly restricted range but extends outside of the Star Lakes to parts of the west branch of the Pearl River.
P. tunguidus has, to date, never been found in the cave. The pond at the mouth of the cave is separated from Star Lakes by a narrow land bar. Currently, the levels of the lake as well as the cave river are controlled by pumps. Historically, floods of the Pearl River may have overflown, bringing lake and cave waters into direct contact. Dry episodes long enough to allow allopatric speciation to proceed are equally possible. That the two Sinodiaptomus share a common ancestor is beyond a reasonable doubt: they are morphologically extremely close and can only be distinguished structurally in males. Females can be distinguished by size: females of ferus are larger than of cavernicolax, but there is a 'grey zone' between them.
P. tunguidus is considered to be endemic to South China [28]. N. schmackeri is widely distributed in the whole of China except the cold, mountainous northwest [29]. Reproductive isolation is supposed to become stronger as genetic distance increases; therefore, barriers should become more hermetic as one moves from the species to the genus level. That is what we seemed to observe, with intergeneric hybrids an order of magnitude more rare than interspecies hybrids.
Could it be that the hybrids are an artifact of human disturbance of the river basins? China is reputed to have constructed some of the first and longest canals in the world, such as the Jing Hang Canal, the 1800 km long Grand Canal linking the Yellow River to the Yangtze basin, built between 486 BC and 610 AD, and the Ling Qu canal in Guangxi Province, between the Yangtze and the Pearl River, built around 214 BC and which is 36 km long. In S. sarsi and the couple S ferus-cavernicolax, another phenomenon seems to occur: here, the ranges are complementary, with possibly a hiatus between the two groups.
The species status of S. cavernicolax, finally, is not in doubt, and neither is its derivation from a common ancestor with S. ferus. Its genetic distance with S. ferus is consistent with a good species, and it appears to have evolved locally in Longyan cave.

Conclusions
In this study, we reconstructed the phylogenetic tree by Bayesian inference and Maximum likelihood method by using COI and ITS sequences from 161 specimens of N. schmackeri, P. tunguidus, S. sarsi, S. ferus and S. cavernicolax. The molecular analysis results provide evidence that there are certain cryptic species in Diaptomidae which have not yet been discovered, and we also find inter-specific and intra-specific hybridization in Diaptomidae with a low hybridization rate. This is the first time that hybrid Diaptomidae species have been found in freshwater.