- Top of page
- Material and Methods
- Conflict of interest
- Author Contributions
- Supporting Information
Ancient lakes, which are defined as those in continuous existence for millions of years, have been recognized as “long-term isolated islands” in terrestrial ecosystems, and exhibit high degrees of biodiversity and endemism (Martens 1997). Numerous evolutionary studies on island systems have been performed due to the abundant intra- and/or interspecific diversity in these small land masses and have provided novel examples of evolutionary patterns (Losos and Ricklefs 2009). As these long-lived lakes are home to landlocked species, the origins and phylogenetic relationships of these endemic species in ancient lakes constitute a biogeographical enigma (e.g., Baikal and Caspian seals, McLaren 1960). On close scrutiny, however, most ancient lakes reveal both steady limnological aging processes and complex histories owing to drastic geological and climatic changes. During the isolation of the predecessors of the present-day lakes, environmental fluctuations (including abiotic and biotic factors) significantly influenced the differentiation of biota between the inland migrants and the original populations (Stager and Johnson 2008). Consequently, fine-scale phylogeographical studies are required to examine patterns of historical geographical fragmentation or gene flow restriction. In particular, application of demographic models of intra- and interspecific diversification to ancient lakes can be used to evaluate evolutionary and/or geological histories of ancient migrants in the basins.
Lake Biwa, a Japanese freshwater lake, harbors many coastal plants that normally inhabit seashores (e.g., Calystegia soldanella [Convolvulaceae], Vitex rotundifolia [Verbenaceae], Lathyrus japonicus [Fabaceae], Arabidopsis kamchatica subsp. kawasakiana [Cruciferae], Raphanus sativus var. raphanistroides [Cruciferae], and Pinus thunbergii [Pinaceae]; Kitamura 1968). This lake, forming approximately 4 million years ago (MYA), is widely recognized as one of the world's few ancient lakes because it is thought to have persisted as a result of the subsidence of a fault (Takaya 1963; Meyers et al. 1993; Kawabe 1994). The coastal plants were postulated to have migrated to the inland lake from coastal populations (Takaya 1963; Kitamura 1968). Some paleogeographic information indicates that Lake Biwa was connected to the Seto Inland Sea at Osaka Bay via a short canal mostly filled with seawater (Seto Inland Sea Basin; Fig. 1; Takaya 1963). Takaya (1963) inferred that populations of the coastal plant Pinus thunbergii (black pine) in Lake Biwa migrated during the middle of the Pleistocene as few paleogeographic fossil records exist (e.g., 1.1–0.4 MYA; Kawabe 1994; Meyers et al. 1993). However, this ancient lake harbors a large number of species (595 animal and 491 plant species; Mori and Miura 1990) with a comparatively low proportion (6%) of endemics (vs. ca. 54% endemics in Lake Baikal and ca. 56% endemics in Lake Tanganyika; Martens 1997). Possibly, this lake has not remained constant over geological time and has repeatedly reconnected with and become isolated from the Seto Inland Sea at Osaka Bay. Lake Biwa has connected with Osaka Bay only via the Seta-Yodo River (Fig. 1); 75 km separates Lake Biwa from Osaka Bay, and the lake is only 85 m above sea level (Ministry of Land, Infrastructure, Transport and Tourism, 2007). Lake Biwa lies in close geographic proximity and shares a related origin and past hydrological connections with the bay, which might result in floral sharing via this river. In addition, sea level fluctuations during the Last Glacial Maximum (LGM; 29.000–19.000 yr bp) and the Holocene are thought to have greatly affected the present distribution of coastal species (Taberlet et al. 1998; Kadereit et al. 2005) (e.g., during the LGM, the eustatic sea level was 125 ± 5 m lower than at present, while during the Holocene [between ca. 7 and ca. 2–1 kyr bp], it was 3–5 m higher than at present [Kevin et al. 1998]). Consequently, coastal plants might have invaded Lake Biwa recently when the sea level rose.
Figure 1. Locations of populations of Lathyrus japonicus in this study. (A) Locations in the Japanese Archipelago and sampling localities in Russia (15) and South Korea (14). (B) Map of Lake Biwa with five extant populations (1–5) with the blue color, Seta-Yodo River, Osaka Plain, and Osaka Bay. Detailed information on these populations is provided in Table S1. The positions where Lake Kawachi and the Ogura-ike Marsh were once located are marked with a broken line with the gray color. The Osaka Plain is represented with the soil color.
Download figure to PowerPoint
Previous phylogeographic studies of L. japonicus (Ohtsuki et al. 2011) and C. soldanella (Noda et al. 2011), found in both Lake Biwa and coastal site, detected unambiguous genetic differentiation between the Lake Biwa's and coastal populations using chloroplast DNA (cpDNA) and nuclear DNA microsatellite (nDNA SSR) loci. Those also detected significantly lower genetic diversity in Lake Biwa's populations than in coastal populations. These results were likely to have been caused by founder events or genetic drift in the inland populations during the long-term isolation of Lake Biwa (Noda et al. 2011; Ohtsuki et al. 2011).
However, given that the Lake Biwa populations harbored a single haplotype of cpDNA sharing with the coastal populations in L. japonicus (Ohtsuki et al. 2011), such a shared haplotype might be interpreted as recent divergence between inland and coastal populations. Because if inland populations have been isolated from coastal populations for long time, they were expected to have unique haplotypes. In addition, the evidence that there are no endemic plant species in of Lake Biwa's lakeshore seems to suggest that the lakeside populations have immigrated from coastal area in recent time and gene flow occurred between inland and seashore populations until recently. Therefore, these previous studies could lead to an incorrect interpretation of the isolation history of Lake Biwa, as few paleogeographic data exist (Takaya 1963). To understand the biogeographic history between coastal and inland lake plants, further studies using multilocus sequencing to estimate the divergence time and population demographic history are needed. Recent advances in population genetics have allowed the estimation of demographic parameters throughout the process of population divergence (e.g., “isolation with migration” model [IM model]: Nielsen and Wakeley 2001; Hey and Nielsen 2004; MiMAR:Becquet and Przeworski 2007).
Lathyrus japonicus (beach pea) is a diploid perennial herb (Fig. 2; Brightmore and White 1963) that commonly occurs in temperate coastal areas of Asia, Europe, and North and South America. Its extensive range is explained by seed dispersal by currents and the ability of the seed to remain viable while floating in seawater for up to 5 years (Brightmore and White 1963). This species is easy to design polymerase chain reaction (PCR) primers and obtain nuclear gene sequences because we can access accumulated expressed sequence tag (EST) information from the Fabaceae. Consequently, beach pea is suitable for elucidating the demographic history of coastal plants that are isolated in Lake Biwa, using multilocus sequencing analyses.
In this study, we applied the “isolation with migration” model (IM model), based on multilocus sequences for the inland and coastal populations of L. japonicus, and: (1) the divergence time of populations of beach pea in Lake Biwa, (2) the historical gene flow between the inland and coastal regions, and (3) the population sizes of the inland and coastal populations. We assessed whether coastal plants colonized inland habitats during the long period of isolation, from 1.1 to 0.4 MYA (Kawabe 1989, 1994; Meyers et al. 1993) or recently, during the LGM. We also examined the level of historical gene flow through the Seta-Yodo River and a founder event with ancestral migrants in Lake Biwa at the time of divergence.