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Hybridization between species or lineages within species has attracted much attention from evolutionary biologists because it helps us to understand the process of speciation (Soltis and Soltis, 2009; Abbott et al., 2013). In contrast, the phenomenon poses serious problems for conservation biologists (Allendorf et al., 2001). Harmful effects of hybridization may lead to the decline or extinction of local populations through two main potential mechanisms (Rhymer and Simberloff, 1996; Todesco et al., 2016). First, if hybrids are sterile or have low fitness, population growth rates may decline below the replacement rates due to wasted reproductive effort; this process is termed demographic swamping (Wolf et al., 2001). Second, if introgression via fertile or semifertile hybrids occurs, one or both parental lineages may be replaced by hybrid progeny with superior fitness; this process is defined as genetic assimilation (Wolf et al., 2001). Because these two processes have an especially large impact on small populations that require an urgent conservation program (Price and Waser, 1979), it is important to grasp the whole picture of ongoing hybridization to develop an efficient conservation strategy.
Rear-edge populations, defined as populations at the low latitudinal margin of species’ distribution (Hampe and Petit, 2005), often deserve high conservation value because they have long-term stores of genetic variation (Provan and Maggs, 2012; Rehm et al., 2015; Evans et al., 2016). Evidence from phylogeographic surveys suggests that rear-edge populations often harbor high genetic diversity and/or uniqueness because they are likely to have persisted under the climate oscillations caused by recent glacial–interglacial cycles during the late Quaternary (Hewitt, 2000, 2004). In addition, they are also thought to acquire local adaptations to the warm and dry conditions with which they have to cope (Granda et al., 2018); these adaptations may be a key for species survival in ongoing global warming. Therefore, rear-edge populations are considered to be important for species conservation from long-term perspectives (Hampe and Petit, 2005).
Ashiu Forest Research Station (hereafter Ashiu), located in central Honshu, Japan (Fig. 1A), is a university field site that comprises a wide range of natural forest. The forest is renowned for its rich temperate flora, with approximately 800 seed plants within the area of ca. 4,200 ha (Yasuda and Nagamasu, 1995), which has attracted the attention of many biologists (Nakai, 1941; Kato and Okuyama, 2004; Sakaguchi et al., 2008; Watanabe, 2016). In particular, Ashiu has been recognized as a phytogeographically important area, because south-westernmost populations of several plant species on Honshu, such as Hemerocallis middendorffii Trautv. et C.A.Mey and Viola brevistipulata (Franch. et Sav.) W.Becker subsp. brevistipulata var. brevistipulata, are located in this forest (Science Museum Net: http://science-net.kahaku.go.jp/?ln=en). Recently, however, these rear-edge populations are at risk of extinction due to deer over-grazing on vegetation, which has become increasingly intense since the early 2000s (Sakaguchi et al., 2008). For these reasons, there is an increasing awareness that Ashiu is an important area with high conservation priority for several species.
Carex podogyna Franch. et Sav. (Cyperaceae) is a sedge plant endemic to Japan, and one of the species which have a rear-edge population in Ashiu (Fig. 1A). In Ashiu, only one small population occurs, in a riparian terrace (ca. 300 m2), which is separated from the closest neighboring population (Tango population; Fig. 1A) by approximately 50 km. Over the last two decades, due to deer over-grazing and frequent river floods (Fig. 1B and 1C, respectively), the number of C. podogyna individuals has rapidly decreased (authors’ unpublished data). As a result, C. podogyna is designated as a critically endangered species (CR) in Kyoto Prefecture, in which Ashiu is located (Kyoto Prefecture Red list 2015: http://www.pref.kyoto.jp/kankyo/rdb/bio/flower.html). Besides, because this species is distributed preferentially in a Japan Sea-side region with heavy snowfall, the risk of loss of climatically suitable habitat will increase more and more due to the decrease of snowfall caused by global warming. Such a risk was predicted for a dwarf bamboo of the sect. Sasa (Tsuyama et al., 2008). For plants in fragile and changing environments, ex-situ conservation is considered to be an efficient conservation strategy in the short, medium, and even long term (Mounce et al., 2017); thus, an urgent ex-situ conservation program is desirable for the rear-edge population of C. podogyna in Ashiu. To establish an ex-situ population for C. podogyna in Ashiu, the genetic variation of the wild population needs to be evaluated and compared with that of other populations to assess its evolutionary potential. In addition, because Carex species have been reported to frequently form interspecific hybrids (Maguilla and Escudero, 2016), it is essential to verify whether hybridization has occurred and, if so, to assess the risk of demographic swamping or genetic assimilation.
The overall goal of this study was to genetically inform the conservation strategy to appropriately establish the ex-situ population of C. podogyna in Ashiu. To achieve this, we performed molecular genetic analysis on wild and ex-situ populations. Specifically, we first evaluated the current genetic diversity of the wild population using microsatellite markers and compared it with that of other populations from other regions. We then tested for the presence of interspecific hybridization and estimated its harmful effect on the survival of the wild population, because there are some closely related species in the vicinity of the wild C. podogyna population that can potentially hybridize with C. podogyna. Finally, we compared the genetic variation of the ex-situ population to that of the wild population in order to determine whether or not genetic variation in the ex-situ population fully reflects that in the parental population.
To investigate the genetic status of the C. podogyna population in Ashiu, leaf samples were collected from the remaining mature individuals (n = 12). In addition, to compare its genetic diversity with that of other populations, 15 and 16 samples were collected from Yamagata (Tohoku; 38°25′N, 140°27′E) and Gifu (Chubu; 36°26′N, 136°49′E) populations, respectively. Leaves were picked from individuals that were at least 2 meters apart, so that possible clones were not collected (Katsuyama, 2005). The collected samples were immediately dried using silica gel and stored in the dark at room temperature. Genomic DNA was extracted from the dried leaf tissue using a slightly modified cetyltrimethylammonium bromide method (Murray and Thompson, 1980) after removing polysaccharides using HEPES buffer (pH 8.0) (Setoguchi and Ohba, 1995).
For establishing the ex-situ population, seeds were collected from these 12 parental individuals of C. podogyna in May, 2018. The collected seeds were sown on watered petri dishes separately for each mother, and then 163 seedlings (10 to 18 per parent) were randomly chosen from those germinated. The established 163 seedlings were used for DNA extraction by the same method as described above.
To evaluate genetic variation of C. podogyna in Ashiu, microsatellite genotyping was performed using five expressed sequence tag–simple sequence repeat (EST–SSR) markers (Table 1) developed for C. angustisquama Franch. (Nagasawa et al., 2018), a related species of C. podogyna in sect. Podogynae Holm. Polymerase chain reaction (PCR) and fragment size determination followed Nagasawa et al. (2018). We note that PCR amplification of Cang_7187 failed for C. shimidzensis Franch. (Table 2), which was therefore analyzed by DNA sequencing as below. To evaluate the remaining genetic variation of the wild population in Ashiu, genetic diversity parameters in terms of the number of alleles (A) and observed and expected heterozygosity (HO and HE, respectively) were calculated using GenAlEx 6.5 (Peakall and Smouse, 2012).
Table 1. Genetic diversity statistics of the five EST-SSR markers used in this study for three populations
| Marker | ASH (N=12) | YMG (N=16) | GIF (N=16) |
|---|
| A | HO | HE | A | HO | HE | A | HO | HE |
|---|
| Cang_4293 | 1 | 0.00 | 0.00 | 5 | 0.53 | 0.62 | 3 | 0.63 | 0.45 |
| Cang_1881 | 1 | 0.00 | 0.00 | 3 | 0.20 | 0.66 | 2 | 0.38 | 0.47 |
| Cang_7187 | 1 | 0.00 | 0.00 | 2 | 0.40 | 0.32 | 3 | 0.19 | 0.61 |
| Cang_18857 | 1 | 0.00 | 0.00 | 3 | 0.33 | 0.52 | 4 | 0.50 | 0.58 |
| Cang_3862 | 1 | 0.00 | 0.00 | 4 | 0.33 | 0.66 | 4 | 0.38 | 0.58 |
| Average | 1.00 | 0.00 | 0.00 | 3.40 | 0.36 | 0.56 | 3.20 | 0.41 | 0.54 |
Note: A = number of alleles per locus; HO = observed heterozygosity; HE = expected heterozygosity.
Table 2. Fragment sizes amplified by five EST–SSR markers developed in
Nagasawa et al. (2018)| Species | POP | Genotype | N | Cang_4293 | Cang_1881 | Cang_7187 | Cang_18857 | Cang_3862 |
|---|
| Allele 1 | Allele 2 | Allele 1 | Allele 2 | Allele 1 | Allele 2 | Allele 1 | Allele 2 | Allele 1 | Allele 2 |
|---|
| Carex podogyna | Parental population | A | 12 | 157 | 157 | 204 | 204 | 234 | 234 | 304 | 304 | 177 | 177 |
| Ex-situ population | A | 150 | 157 | 157 | 204 | 204 | 234 | 234 | 304 | 304 | 177 | 177 |
| | B | 8 | 149 | 157 | 204 | 222 | 234 | 236 | 304 | 309 | 169 | 177 |
| | C | 3 | 151 | 157 | 204 | 222 | 234 | 236 | 304 | 309 | 169 | 177 |
| | D | 1 | 149 | 157 | 204 | 222 | 234 | 236 | 304 | 311 | 169 | 177 |
| | E | 1 | 149 | 157 | 204 | 222 | 234 | 236 | 304 | 309 | 169 | 177 |
| Carex curvicollis | | | 1 | 149 | 149 | 220 | 222 | 236 | 236 | 309 | 311 | 169 | 169 |
| | | 1 | 147 | 147 | 218 | 218 | 236 | 236 | 309 | 309 | 169 | 169 |
| Carex shimidzensis | | | 1 | 161 | 161 | 222 | 228 | – | – | 309 | 309 | 175 | 175 |
Note: bold numerals mark alleles not detected in the parental C. podogyna population; –, no amplification.
When alleles undetected in the parental population of C. podogyna were observed in seedlings, we considered them as alleles that originated from other Carex species. Carex podogyna is known to naturally hybridize with several related species (Katsuyama, 2005), among which C. curvicollis and C. shimidzensis are the only two species that have been reported to co-distribute in Ashiu (Yasuda and Nagamasu, 1995); therefore, we hypothesized that C. podogyna had hybridized with C. curvicollis or C. shimidzensis. To test this, DNA sequencing analysis was performed using three individuals of C. podogyna from the parental population, two individuals of C. curvicollis collected from near the C. podogyna population, one individual of C. shimidzensis from Ashiu, and 13 putative hybrid individuals from the ex-situ population (Table 2). To determine whether individuals with unexpected genotypes were hybrids between C. podogyna and C. curvicollis or C. shimidzensis, the external transcribed spacer (ETS) region was amplified with primers ETS 1f and 18S-R (Starr et al., 2003). PCR was conducted following the protocol of Starr et al. (2003), and the PCR products were sequenced following Takahashi and Setoguchi (2018). The obtained DNA sequences were edited and assembled using BioEdit 7.2.5 (Hall, 1999). Heterozygous sites were called out when the peak height ratio of a secondary peak to a primary peak exceeded 50%. Sequence alignment was performed with ClustalW (Thompson et al., 1994) with default settings. All sequences were deposited in the DNA Data Bank of Japan (DDBJ), and accession numbers of the sequences used in this study are listed in Table 2.
The microsatellite genotyping rate was 100% for the Ashiu, Yamagata and Gifu populations, and then five, 17 and 16 scorable alleles were detected in each population, respectively (Table 1). While no identical genotypes were found within the Yamagata and Gifu populations, all individuals in Ashiu showed the same genotype with all six loci fixed to homozygotes (genotype A; Table 2). In agreement with this result, genetic diversity in terms of HO and HE showed a complete loss of genetic diversity in Ashiu, while the Yamagata and Gifu populations harbored substantial genetic variation (Table 1): mean values of HO and HE were 0.36 and 0.56 for Yamagata and 0.41 and 0.54 for Gifu, levels which were similar to those of closely related Carex species, such as C. doenitzii (HO = 0.45, HE = 0.59: Nagasawa et al., 2020) and C. otayae (HO = 0.40, HE = 0.36: Nagasawa et al., 2021). This result indicated that the lack of genetic variation detected in the Ashiu population is unlikely to be due to a lower informativeness of the C. angustisquama-derived genetic markers for C. podogyna. In addition, given the same sampling strategy for the three populations and the presence of seedlings in the wild Ashiu population (S. F., personal observation), we do not consider that possible clonal reproduction of C. podogyna would have resulted in genetic purity in the Ashiu population. Instead, we propose that the rear-edge population in Ashiu lost genetic diversity via strong bottlenecks and/or genetic drift due to isolation, as a small population, from other populations in central distribution areas. Further study is required to reveal whether the observed low genetic diversity in the Ashiu population is a phenomenon specific to rear-edge populations or is also found in other regions, by analyzing more population samples with genetic markers.
For the ex-situ population, the microsatellite genotyping rate was 99% and 12 scorable alleles were detected. Although genetic theory predicts that all 163 individuals should share the same genotype as inherited from parental individuals, four new genotypes (genotypes B–E; Table 2), in which the alleles were undetected in parental individuals of C. podogyna, were found in 13 individuals (Table 2). Because some of these alleles were not found in other populations of C. podogyna (authors’ unpublished data from a range-wide phylogeographic study), and consistent with being heterozygotes comprising parental alleles of C. podogyna and C. curvicollis (Table 2), we hypothesized that these alleles arose via natural hybridization with C. curvicollis.
To test this hypothesis, 19 ETS sequences with lengths ranging from 431 bp to 576 bp were obtained. After removing sequence gaps, a sequence alignment of 431 bp long was obtained. The aligned sequences harbored 19 segregating sites between C. podogyna and C. curvicollis, and 20 segregating sites between C. podogyna and C. shimidzensis (Table 3). The putative hybrid individuals harbored 17 heterozygous sites, at which each allele matched one or other of the species-specific alleles of C. podogyna and C. curvicollis (Table 3). Note that the DNA sequences of C. podogyna, C. curvicollis and putative hybrids were identical within each species/population; that is, they were fixed to three distinct sequences. This pattern of DNA sequence variation indicates that the individuals with unexpected genotypes likely originated from natural hybridization between C. podogyna and C. curvicollis. Because C. curvicollis is a common riparian species in Japan, both species have been found in Ashiu to occur so close to each other that pollen flow can occur. Although a natural hybrid between these two species is known as C. ×hosoii T. Koyama, this hybrid has never been recorded in Ashiu (Yasuda and Nagamasu, 1995), and actually, we found no hybrid genotype in mature individuals that were sampled from the natural habitat in Ashiu (Table 2). These facts suggest that C. ×hosoii cannot survive in the natural environment for some reason. Our observation of these hybrid individuals during cultivation provided a possible explanation for the lethality of hybrids in natural environment: most of them formed variegated leaves in the early stage of growth (Fig. 1D). Since variegated leaves are known to inhibit plant growth via impaired photosynthesis (Funayama and Terashima, 1999; Funayama et al., 2001), C. ×hosoii may be selected against in the natural environment due to its forming variegated leaves, which should be less productive in terms of photosynthesis. Other genetic and ecological factors, including possible hybrid sterility (Koyama, 1956) and extrinsic ecological isolation (as microhabitat segregation is apparent between the two Carex species), may be equally influential, and thus need to be examined in future studies.
Table 3. Segregated sites among
C. podogyna,
C. curvicollis and
C. shimidzensis| Species | N | Variable site positions in ETS alignment from forward primer (bp) |
|---|
| 3 | 11 | 13 | 30 | 37 | 83 | 84 | 86 | 103 | 115 | 134 | 150 | 153 | 156 | 166 | 178 | 185 | 203 | 235 | 236 |
|---|
| Carex podogyna | 3 | C | T | T | C | C | A | C | C | A | G | T | T | C | T | T | T | T | A | A | A |
| Carex curvicollis | 2 | T | – | – | – | – | – | G | – | T | – | C | C | – | – | – | C | C / T | G | G | C |
| Carex shimidzensis | 1 | – | C | C | T | G | T | G | C | T | C | – | C | T | G | C | C | C | – | G | T |
| Putative hybrids | 13 | C / T | – | – | – | – | – | C / G | – | A / T | – | T / C | T / C | – | – | – | T / C | C / T | A / G | A / G | A / C |
| Species | Variable site positions in ETS alignment from forward primer (bp) | GenBank accession | Voucher specimen accession |
|---|
| 240 | 247 | 261 | 266 | 317 | 323 | 326 | 370 | 398 | 437 |
|---|
| Carex podogyna | A | C | G | C | C | A | T | A | T | C | LC570900–LC570902 | KYO 00023452 |
| Carex curvicollis | T | C / T | A / G | G | – | G | C | T | C | A / C | LC570903, LC570904 | KYO 00025853 |
| Carex shimidzensis | – | T | – | – | T | – | – | T | – | – | | |
| Putative hybrids | A / T | – | – | C / G | – | A / G | C / T | A / T | T / C | A / C | LC570905–LC570917 | KYO 00025856 |
Note: voucher specimens are deposited at the herbarium of Kyoto University; –, the allele is the same as that of C. podogyna.
Our genotypic data revealed that the Ashiu population of C. podogyna has experienced a loss of genetic diversity, as well as a severe reduction of effective population size (Aguilar et al., 2008; Cho et al., 2020). Our results also showed that despite some cryptic natural hybridization, the rear-edge C. podogyna population still maintained genetic purity, indicating that the C. ×hosoii in Ashiu does not contribute to genetic introgression, possibly leading to genetic assimilation (Wolf et al., 2001). Nevertheless, the hybridization is likely to reduce the population growth rate due to the wasted reproductive effort, as detected in our seeding experiment in which 8% (13/163) of the seedlings were identified as hybrids. From the above results, together with the increasing risk of loss of suitable habitat due to ongoing global warming, we propose that ex-situ conservation is an effective way to safely preserve the remaining genetic diversity of C. podogyna in Ashiu as a potential genetic resource for species conservation, although there is little risk of genetic assimilation in the natural environment. Finally, our results emphasize the importance of genetic verification to determine whether an ex-situ population consists of genetically pure individuals, even when hybridization is not observed in the natural environment of the source population.
