Aim: The evolutionary importance of paleoclimate regimes has been noted in biogeographic studies. However, little is known about how paleoclimate differences shaped the biogeographic pattern and diversification history of the freshwater fauna in important zoogeographical boundary regions. Here, we aim to investigate how past regional climatic differences have shaped the biogeographic history of the inland aquatic fauna in China using an endemic freshwater crab species complex found on both sides of the Qinling Mountains–Huaihe River Line (QHL), a critical ecological boundary in eastern China, as a model system.

Location: Eastern China, the Qinling Mountains–Huaihe River Line.

Taxon: The Sinopotamon yangtsekiense species complex.

Methods: A total of 482 individuals of Sinopotamon yangtsekiense sensu lato were collected from 34 localities throughout its entire distributional range. The phylogeographic analyses of population structure, morphological and genetic variations, and demographic dynamics were made based on multiple mtDNA and nuDNA loci and on morphological traits. Fine-tuned ecological niche modeling was used to reconstruct the location of climatically suitable areas that existed during the Last Glacial Maximum.

Results: The divergence of two freshwater crab lineages across the QHL correlated with significant past variations in monsoon intensity and with the location of multiple refuges. The divergence time was broadly consistent with the timing of the critical paleoclimate transition event in the mid-Pleistocene (95% HPD, 0.48–1.06 Ma). Each freshwater crab lineage has evolved distinct male genital traits associated with their isolation in areas with different precipitation rates and temperatures in the past. The patterns of crab distribution observed today reflect past contractions of the two lineages in response to glacial and interglacial cycles during the Pleistocene, followed by their subsequent rapid expansion after the Last Glacial Maximum (~15 kya).

Main conclusions: Populations of the widespread species Sinopotamon yangtsekiense s.l. experienced a deep division in the past that led to the phylogeographical isolation observed today. The two main drivers of genetic isolation in this taxon were (a) differences in the intensity of the monsoons on each side of the QHL boundary during the mid-Pleistocene, and (b) isolation of different populations of S. yangtsekiense s.l. in a number of separate refuges during the LGM.

Eight microsatellite loci primers developed by Sun et al. (2009) were used for PCR amplification: hxx3, hxx6, hxx7, hxx9, Ga2, Gaca12, Gagt21, and Gagt12 (see Table S2 in Supporting Information). Amplification conditions are described in Sun et al. (2009). PCR products were separated on 6.5% polyacrylamide gels using a LI-COR 4300 automated DNA sequencer and analyzed using LI-COR SAGAGT software.

The first (G1) and second (G2) gonopods of male brachyurans are typically complex and exhibit considerable morphological variation among taxa. This is especially true in freshwater crabs where the unique shape of the G1 is used as a high-weight species-level character in taxonomic identification, along with characters of the G2, and the sixth pleonal segment (A6). We focused on the morphology of the G1, G2, and A6 of males because of difficulties quantifying the characters of the female genitalia. Measurements of the subterminal segments (SS) of the G1 and G2 were made along a straight line beginning at the midpoint of the basal margin and ending at the midpoint of the distal margin (at the junction between the two parts). Measurements of the terminal articles (TAs) of the G1 were made on the ventral side along the midline beginning at the midpoint of the junction between the TA basal margin and the distal SS, and ending at the TA tip. We measured five morphological traits from 147 adult male specimens belonging to 27 populations as follows: (a) the G1 SS and G1 TA; (b) the G2 SS and G2 TA; (c) the height of the G1TA ventral (V) and dorsal (D) lobes; (d) the width (W) and length (L) of the sixth pleonal segment (A6); (e) and the angle of the G1TA with respect to the longitudinal axis of the G1 and SS (G1 TA angle). G1 TA used the average of the V and D. These values were converted into ratios (except the G1 TA angle) to exclude errors due to age and size as follows: G1 TA/SS, G2 TA/SS, G1 TAV/D, and A6 W/L. Coefficients of variation (CV%) were determined to indicate morphological variability.