Characterization of 39 microsatellite markers from Nuphar shimadai (Nymphaeaceae) and cross-amplification in two related taxa.

PREMISE OF THE STUDY: Microsatellite loci were developed for Nuphar shimadai (Nymphaeaceae) to evaluate the population genetic dynamics for conservation purposes. The species is an endemic aquatic species in Taiwan that is endangered by anthropogenic activities. METHODS AND
RESULTS: A magnetic bead enrichment protocol was used to identify 72 potential microsatellite loci and develop 39 microsatellite markers from N. shimadai. The number of alleles per locus ranged from one to 10 per locus, with levels of observed heterozygosity ranging from 0 to 1.0 within populations. As a result of inbreeding within isolated populations, 65% of loci significantly deviated from Hardy-Weinberg equilibrium within populations.
CONCLUSIONS: These novel markers should be valuable tools to evaluate the genetic diversity within the endangered aquatic taxon N. shimadai for conservation and reintroduction purposes in Taiwan.

The genus Nuphar Sm. (Nymphaeaceae) contains 52 extant aquatic taxa, which are widely distributed in lowland lakes and ponds in the temperate Northern Hemisphere, including Europe, North America, and Asia (Padgett et al., 1999). Nuphar shimadai Hayata, known as yellow water lily, is a Taiwanese endemic aquatic species that typically occurs in lowland lakes and ponds of northern Taiwan. Remaining populations of N. shimadai are rare due to the severe destruction of natural habitat. As a result, N. shimadai is recognized as a Critically Endangered species under the IUCN Red List Categories and Criteria Version 3.1 (IUCN, 2012) and listed in A Preliminary Red List of Taiwanese Vascular Plants (Wang et al., 2012). Therefore, it is urgent to explore and identify microsatellite markers, which can be used to effectively evaluate genetic diversity in N. shimadai. Previous studies of Nuphar only focused on matK, ITS regions of nuclear ribosomal DNA (Padgett et al., 1999), and AFLPs (Shiga et al., 2006); these markers are used primarily for phylogenetic studies, and thus do not have the resolution to address population‐specific questions. Moreover, microsatellite markers have only been developed for N. lutea (L.) Sm. (Ouborg et al., 2000) and N. japonica DC. (Kondo et al., 2016), and the cross‐amplification and polymorphism of these markers to N. shimadai has been shown to be limited in preliminary tests. To date, highly polymorphic DNA regions have scarcely been used to elucidate intraspecies relationships of Nuphar species. In the present study, we developed 39 highly variable microsatellite primer pairs from N. shimadai and examined their applicability in two related Nuphar species. These polymorphic microsatellite markers will be a viable strategy to evaluate population genetics and demographic history of N. shimadai and its related taxa, which can be used to infer inter‐ and intraspecific relationships. Therefore, these markers can also assist in the establishment of conservation management strategies for N. shimadai and related species.

METHODS AND RESULTS

Sampling and DNA extraction

Five populations of N. shimadai from throughout its geographic distribution range were sampled from different ponds in two localities in northern Taiwan (Taoyuan: populations WP, GPa, GPb, GPn, and Ilan: population LD; Appendix 1). Nuphar pumila (Timm) DC. subsp. sinensis (Hand.‐Mazz.) Padgett was obtained from Jiangxi Province in China (population GJ); N. japonica was acquired from Owase, Japan (population JP) (Appendix 1). All voucher specimens are deposited in the Herbarium of the Taiwan Forestry Research Institute (TAIF). Genomic DNA was extracted from silica gel–dried leaf tissue using a Genomic DNA Extraction Kit (RBC Bioscience, Taipei, Taiwan).

Development of microsatellite markers

The microsatellite‐enriched library of N. shimadai was constructed using a magnetic bead procedure (Liao et al., 2009; Chiang et al., 2011). The genomic DNA of N. shimadai was digested by the restriction enzyme MseI (Promega Corporation, Madison, Wisconsin, USA). Fragments from 400 to 1000 bp were isolated and ligated to the double‐stranded adapter (complementary oligo A: 5′‐TACTCAGGACTCAT‐3′; phosphorylated oligo B: 5′‐GACGATGAGTCCTGAG‐3′). To enrich the partial genomic library, the adapter‐specific MseI‐N primer (5′‐GATGAGTCCTGAGTAAN‐3′) was used to perform 18 cycles of prehybridization PCR amplification. The PCR protocol was as follows: initial denaturation at 94°C for 5 min, followed by 18 cycles of 94°C for 30 s, 53°C for 1 min, and 72°C for 1 min. PCR products were isolated, denatured, and separately hybridized to two biotinylated probes (B‐(AG)15, B‐(AC)15) at 68°C for 1 h. One milligram of Streptavidin MagneSphere Paramagnetic Particles (Promega Corporation) was added to the hybridized PCR products to capture the hybridizations at 42°C for 2 h. The enriched (AG) and (AC) DNA fragments were washed with high‐ and low‐salt solutions, then eluted, and purified DNA fragments were used as templates for 25 cycles of PCR amplification using MseI‐N. Purified DNA fragments were cloned using the pGEM‐T Easy Vector System (Promega Corporation) and screened by PCR (primer pairs: (AG)10 or (AC)10 and SP6 or T7). A total of 239 selected clones were isolated and sequenced in both directions using the ABI BigDye Terminator 3.1 Cycle Sequencing Kit with the ABI PRISM 3700 DNA Sequencer (Applied Biosystems, Waltham, Massachusetts, USA). Microsatellite loci were identified applying the Tandem Repeats Finder version 4.07b (Benson, 1999). A total of 72 primer pairs were developed using FastPCR software version 6.4.18 (Kalendar et al., 2011) based on the following detailed limitation settings: amplicon size range from 100 to 350 bp, an optimal annealing temperature of 55°C, and a GC content ranging from 40–60%. The optimal annealing temperature for each locus was obtained from temperature‐gradient PCR under the following PCR conditions: 94°C for 2 min; 35 cycles of 94°C for 45 s, 50–60°C gradient for 45 s, and 72°C for 50 s; followed by a final elongation at 72°C for 7 min. The PCR reaction volume per 20 μL contained approximately 5 ng of genomic DNA, 4 μL of 5× reaction buffer, 0.2 μM dNTP mix, 2 mM MgCl2, 0.5 units of GoTaq MDx Hot Start Polymerase (Promega Corporation), 0.2 μM of both primers, and sterile ddH2O to total 20 μL. PCR amplification was performed on a Labnet MultiGene 96‐well Gradient Thermal Cycler (Labnet, Edison, New Jersey, USA). From the 72 microsatellite loci, we obtained 39 primer pairs with discrete amplicon peaks, and the target PCR products of these 39 microsatellite loci were electrophoresed on 1% agarose gel and confirmed by sequencing. To evaluate the genetic diversity for N. shimadai and two related taxa, microsatellite loci were amplified by PCR and the target amplicons were visualized using the FloGel FGIS‐3 fluorescent gel image system (Top BIO Co., Taipei, Taiwan) after being re‐resolved on a 10% polyacrylamide gel. Amplicon molecular weights containing microsatellite loci were scored using Quantity One version 4.6.2 software (Bio‐Rad Laboratories, Hercules, California, USA) and adjusted manually.

Molecular data analysis and results

Genetic diversity parameters, including average and effective numbers of alleles per locus, expected and observed heterozygosity (H e and H o), and deviation from Hardy–Weinberg equilibrium, were evaluated using GenAlEx version 6.5 (Peakall and Smouse, 2012). Microsatellite sequences were submitted to GenBank, including the 39 microsatellite markers developed and used in this study (Table 1) and an additional 33 monomorphic microsatellite markers that were developed but not used (Appendix 2). The numbers of observed and effective alleles per locus ranged from one to 10 and one to 4.6, respectively. Levels of H o and H e varied from 0 to 1.0 and 0 to 0.8, respectively. A total of 30, 23, 27, 33, and 39 loci deviated significantly from Hardy–Weinberg equilibrium in the WP, GPb, GPa, GPn, and LD populations, respectively (P < 0.05; Table 2). Tests for linkage disequilibrium between loci were performed and no pairs of loci exhibited significant linkage disequilibrium.

Table 1

Characteristics of 39 microsatellite loci developed in Nuphar shimadai

Locus	Primer sequences (5′–3′)	Repeat motif	Allele size range (bp)	T a (°C)	GenBank accession no.
NS‐AG‐144	F: CACTGCTGTGTTACAGGAAG	(AG)10	103–107	50	MH396370
	R: TCATGGCATTAGCATCTAGG
NS‐AG‐146	F: TTGGTGATGCTTATAACACG	(TG)14	214–232	50	MH396371
	R: GTACATACAATCTCTCAAGG
NS‐AG‐152	F: TGTGATACAATCTAGTGTCC	(TC)14	172–212	52	MH396372
	R: TTTCTGAATCACTCTAGTGG
NS‐AG‐158	F: AGCATCTGTAAGATGTACGC	(TG)9	157–217	56	MH396373
	R: TGTGCTATCAATGCATTGCC
NS‐AG‐159	F: GGTCAATGAGAGTTTGTAGG	(TG)9	174–180	56	MH396374
	R: CATATGTTTCCCTCGTGCAC
NS‐AG‐164	F: ATGGCATCATAGGATAAGCC	(TG)14(AG)6	186–188	57	MH396375
	R: TCCAGCAATTTCACGCTTGC
NS‐AG‐167	F: CTCATCACATGGAGGGAATC	(CA)11(TACA)14	256–300	55	MH396376
	R: TGGAGATCCTGACCAATTCC
NS‐AC‐152	F: TCGACCTATTTGGTTTGACC	(CA)11	182–216	57	MH396377
	R TGGTATGGATCTGGTCAGAC
NS‐AG‐196	F: ACTAGACTGTGACATACCTG	(TG)10	158–190	57	MH396378
	R: TGAATCATCGCATGTCCTGG
NS‐AG‐207	F: TCTTTGAGACATGGTACCTG	(TG)10	168–182	56	MH396379
	R: CCATACAAACGTCAATTCAC
NS‐AG‐223	F: AGTGACAGAGTCATAGGTAC	(AC)8	202–210	56	MH396380
	R: TAGGGCTTAGACAATGGACC
NS‐AG‐224	F: GAACCTTCACAGTGAAACAG	(TG)9N(AG)10	270–280	56	MH396381
	R: TTTCAAACATGCTGCCAAGC
NS‐AG‐225	F: AGCCAAAGTTCTTACCATCG	(TC)15	230–250	55	MH396382
	R: CTAGATTTGGACCGTACAAG
NS‐AC‐139	F: AAGCCTTCCGAATTCAGAAG	(AG)12	150–162	56	MH396383
	R: GCCAACTTATGAATGGAAGT
NS‐AC‐143	F: ACATGGTGTTGTAGCTAGGC	(AG)22	218–242	57	MH396384
	R: GCTGCACTACTTGGCTTCAC
NS‐AC‐149	F: CTTGCTTGCGCTAGGTGTTG	(CT)13	276–312	58	MH396385
	R: CTATGTGACAGGGACTCTGC
NS‐AC‐150	F: TTGGATGCACGGGCTTATAG	(CT)10	204–218	58	MH396386
	R: CTGTGCTTGTCACAATGATCC
NS‐AC‐155	F: AAAACTACCACCCAAGGGAG	(TC)23	202–226	58	MH396387
	R: CATCTCTTCTTCTCCATGTG
NS‐AC‐165	F: TGTGAATCAACAAGAGGAAG	(AG)12	164–180	57	MH396388
	R: ACTTGGATGGGGATTCTTAC
NS‐AC‐170	F: CACCATAGCATACCCATGTG	(GA)9	144–156	57	MH396389
	R: ATCATTCGTTCGACAACTGC
NS‐AC‐171	F: GTCTTGCTTATGAAGGTAGG	(TC)14	182–224	56	MH396390
	R: AGTAGAATCAGCATACGTGC
NS‐AC‐172	F: TGAGCTTCTCCCCAAGATTG	(AC)11	140–146	56	MH396391
	R: GTTTCATTTCTGCAGCAGAG
NS‐AC‐176	F: GTGGTAATACAGGAGCCAGC	(TG)8	208–234	58	MH396392
	R: GGAGTGCCCATTGACATATC
NS‐AC‐178	F: ATATAGGAAGCCTGCCGAAC	(GAGTGT)3	138–142	53	MH396393
	R: GGCACCATTAGCTCTGATAC
NS‐AC‐182a	F: GACATGCACATTGCAACAGAG	(AG)17	186–206	56	MH396394
	R: CAAGCGGCTGTCTAATGTTC
NS‐AC‐182b	F: GAACATTAGACAGCCGCTTG	(AG)19	180–184	57	MH396395
	R: TTAGGCCGTAGGCGTTCAAC
NS‐AC‐186	F: GATCTTTTGTGTTACGTGAG	(GA)15	122–126	57	MH396396
	R: GAGAATTTGTTGCATGCACG
NS‐AC‐189	F: CAAAGCCAGCCAAAGTAACG	(TG)4(GA)29	170–196	56	MH396397
	R: TTCTCTTTCATCCCCATCAC
NS‐AC‐198b	F: AACAAAGCCTGCAATTTGTG	(AG)11	160–190	57	MH396398
	R: ACGCACATGAAGATACTTGC
NS‐AC‐206	F: TCTTCAAATCCAATGGTTCG	(GA)14	122–134	57	MH396399
	R: TAAAGGTACAACCACAGTCC
NS‐AC‐136	F: CGTGGTCTACCAAAAACTAG	(TG)4(AG)12	170–192	57	MH396400
	R: ACTGCCCAAAATCTTCGATC
NS‐AC‐111	F: GCATCAGGAAGCACACCATC	(TC)13	132–136	58	MH396401
	R: GTCGTACAGGAATGGAGACG
NS‐AC‐112	F: CCACTAGAAGAATCCGATCC	(AG)18	136–140	56	MH396402
	R: AAGGGTACTGACCTACCAAG
NS‐AC‐129	F: CTAACCATAAACCACTGCTG	(TC)18	174–176	53	MH396403
	R: TCTTAGAAAACCCACTTCCC
NS‐AC‐130	F: CACACTTAACAACTACCACC	(TC)27	164–176	55	MH396404
	R: TCTTGAGTCATCGAATTGTC
NS‐AC‐131	F: TGGAAGAAGTTGCTGTGATG	(AG)10	174–176	56	MH396405
	R: GACCTAAGTTAGCAAATACC
NS‐AC‐132	F: TGTCCTCATGATATGTCAAC	(TG)14	164–204	55	MH396406
	R: GTGAACACATTTACAGTGTC
NS‐AG‐135	F: GGAAACGATCTCCGGAAACG	(CT)16	164–168	58	MH396407
	R: GGCAATATTCCGAGTCGTAC
NS‐AC‐173	F: CAAGCTTGCATTTGCATCTC	(CA)6N(AG)19	274–280	53	MH396408
	R: TAATCCCAGCCATCCAATGG

T a = annealing temperature.

Table 2

Genetic variation of 39 novel microsatellite loci in five populations of Nuphar shimadai.a

Locus	WP (n = 22)				GPb (n = 13)				GPa (n = 11)				GPn (n = 16)				LD (n = 78)
	A	A e	H o	H e b	A	A e	H o	H e b	A	A e	H o	H e b	A	A e	H o	H e b	A	A e	H o	H e b
AG144	2	1.3	0.0	0.2***	2	1.7	0.0	0.4***	2	1.2	0.0	0.2***	2	2.0	0.0	0.5***	4	3.6	0.7	0.7ns
AG146	2	1.3	0.0	0.2***	2	1.2	0.0	0.1***	2	1.2	0.0	0.2***	2	2.0	0.0	0.5***	4	2.9	0.6	0.7ns
AG152	2	1.2	0.0	0.2***	3	2.3	0.0	0.6***	2	1.7	0.0	0.4***	4	2.2	0.0	0.5***	6	3.8	0.8	0.7ns
AG158	6	3.3	0.5	0.7ns	2	2.0	1.0	0.5***	4	2.4	1.0	0.6***	4	2.9	1.0	0.7***	3	1.9	0.0	0.5***
AG159	3	1.2	0.0	0.2***	2	1.2	0.0	0.1***	2	1.2	0.0	0.2***	3	1.5	0.0	0.3***	4	2.9	0.7	0.7ns
AG164	2	1.4	0.0	0.3***	2	1.2	0.0	0.1***	2	1.2	0.0	0.2***	2	1.8	0.0	0.4***	4	2.3	0.7	0.6ns
AG167	3	2.3	0.5	0.6ns	4	2.4	0.3	0.6**	2	1.7	0.0	0.4***	2	1.4	0.0	0.3***	4	3.0	0.8	0.7***
AC152	4	2.7	0.4	0.6ns	4	3.5	1.0	0.7***	2	2.0	1.0	0.5***	3	2.3	0.4	0.6ns	3	1.5	0.0	0.4***
AG196	6	2.8	0.9	0.6***	4	3.5	1.0	0.7***	4	2.8	1.0	0.7***	5	2.5	0.8	0.6ns	4	1.4	0.1	0.3*
AG207	1	1.0	0.0	0.0	2	1.2	0.0	0.1***	1	1.0	0.0	0.0	2	1.8	0.0	0.4***	3	1.8	0.0	0.5***
AG223	1	1.0	0.0	0.0	1	1.0	0.0	0.0	2	1.4	0.0	0.3***	2	2.0	0.0	0.5***	3	1.5	0.0	0.4***
AG224	1	1.0	0.0	0.0	1	1.0	0.0	0.0	1	1.0	0.0	0.0	2	1.1	0.0	0.1***	4	1.2	0.0	0.2***
AG225	5	3.8	0.0	0.7***	2	1.7	0.0	0.4***	1	1.0	0.0	0.0	3	1.7	0.0	0.4***	6	3.6	0.8	0.7ns
AC139	4	2.2	1.0	0.5***	4	2.3	1.0	0.6***	2	2.0	1.0	0.5***	3	2.5	0.8	0.6ns	4	3.2	0.5	0.7ns
AC143	3	1.7	0.5	0.4ns	4	2.3	1.0	0.6***	4	2.4	1.0	0.6***	7	3.4	0.8	0.7ns	4	2.0	0.1	0.5***
AC149	4	2.1	0.5	0.5ns	2	2.0	0.8	0.5*	4	1.9	0.5	0.5ns	4	2.4	0.8	0.6ns	10	4.4	0.7	0.8ns
AC150	2	1.9	0.0	0.5***	1	1.0	0.0	0.0	3	1.5	0.0	0.3***	2	1.1	0.0	0.1***	6	3.2	0.7	0.7ns
AC155	3	2.2	0.8	0.5***	3	2.1	0.6	0.5ns	2	1.9	0.8	0.5*	5	2.6	0.3	0.6**	5	1.8	0.0	0.4***
AC165	4	2.7	0.5	0.6ns	2	2.0	0.0	0.5***	2	1.2	0.0	0.2***	2	1.8	0.7	0.5*	4	2.5	0.0	0.6***
AC170	2	1.2	0.2	0.2ns	2	1.4	0.0	0.3***	2	1.4	0.0	0.3***	2	1.1	0.0	0.1***	5	3.4	0.9	0.7ns
AC171	5	2.5	0.7	0.6ns	2	1.3	0.2	0.2ns	2	1.7	0.5	0.4ns	1	1.0	0.0	0.0	8	3.7	0.1	0.7***
AC172	2	1.7	0.5	0.4ns	1	1.0	0.0	0.0	1	1.0	0.0	0.0	3	1.8	0.6	0.4ns	6	4.5	0.6	0.8*
AC176	2	1.5	0.4	0.3ns	2	1.9	0.0	0.5***	2	2.0	0.0	0.5***	2	2.0	0.0	0.5***	4	2.3	0.2	0.6***
AC178	2	1.5	0.0	0.4***	2	1.7	0.0	0.4***	2	1.4	0.0	0.3***	2	2.0	0.0	0.5***	4	1.9	0.6	0.5ns
AC182a	3	1.7	0.5	0.4ns	1	1.0	0.0	0.0	1	1.0	0.0	0.0	2	1.3	0.0	0.2***	3	1.2	0.0	0.2***
AC182b	3	1.5	0.0	0.3***	1	1.0	0.0	0.0	2	1.2	0.0	0.2***	2	1.1	0.0	0.1***	2	1.1	0.0	0.1***
AC186	2	1.7	0.0	0.4***	1	1.0	0.0	0.0	2	1.2	0.0	0.2***	3	1.7	0.3	0.4ns	2	1.9	0.7	0.5*
AC189	3	2.9	0.0	0.7***	1	1.0	0.0	0.0	1	1.0	0.0	0.0	2	1.3	0.0	0.2***	5	1.8	0.0	0.5***
AC198b	3	2.9	0.6	0.7ns	1	1.0	0.0	0.0	2	1.7	0.0	0.4***	2	1.4	0.0	0.3***	3	1.3	0.0	0.2***
AC206	4	2.0	0.0	0.5***	2	2.0	1.0	0.5***	2	2.0	1.0	0.5***	3	1.7	0.4	0.4ns	4	1.7	0.1	0.4***
AC136	2	2.0	0.0	0.5***	2	2.0	0.0	0.5***	1	1.0	0.0	0.0	1	1.0	0.0	0.0	4	2.0	0.8	0.5**
AC111	2	1.9	0.0	0.5***	1	1.0	0.0	0.0	1	1.0	0.0	0.0	2	1.9	0.0	0.5***	2	1.4	0.0	0.3***
AC112	2	1.7	0.0	0.4***	1	1.0	0.0	0.0	1	1.0	0.0	0.0	2	1.1	0.0	0.1***	5	2.7	0.8	0.6ns
AC129	2	1.7	0.0	0.4***	1	1.0	0.0	0.0	1	1.0	0.0	0.0	2	1.4	0.0	0.3***	2	1.5	0.0	0.3***
AC130	2	1.7	0.5	0.4ns	1	1.0	0.0	0.0	2	1.9	0.0	0.5***	3	1.8	0.4	0.4ns	2	1.2	0.0	0.1***
AC131	1	1.0	0.0	0.0	1	1.0	0.0	0.0	1	1.0	0.0	0.0	2	1.1	0.0	0.1***	3	1.5	0.0	0.4***
AC132	6	4.4	0.8	0.8ns	4	3.4	0.6	0.7ns	6	4.6	1.0	0.8***	6	4.5	0.6	0.8ns	5	1.4	0.0	0.3***
AG135	2	1.8	0.0	0.4***	2	1.2	0.0	0.1***	3	1.8	0.0	0.4***	3	1.9	0.0	0.5***	5	2.7	0.0	0.6***
AC173	1	1.0	0.0	0.0	1	1.0	0.0	0.0	2	1.2	0.0	0.2***	3	1.1	0.1	0.1ns	5	3.8	1.0	0.7***
Mean	2.8	1.9	0.3	0.4	2.0	1.6	0.2	0.3	2.1	1.6	0.2	0.3	2.7	1.8	0.2	0.4	4.2	2.4	0.3	0.5

A = number of alleles; A e = number of effective alleles; H e = expected heterozygosity; H o = observed heterozygosity; n = number of individuals sampled.

aVoucher and locality information are provided in Appendix 1.

bDeviation from Hardy–Weinberg equilibrium: *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant.

All 39 microsatellite markers were cross‐amplified to N. pumila subsp. sinensis and N. japonica under the same conditions described above, except for annealing procedures displaced by optimal annealing temperature (Table 1). For N. pumila subsp. sinensis and N. japonica, the highest numbers of alleles per locus were four and nine, respectively, and the highest numbers of effective alleles were 3.8 and 6.8, respectively (Table 3). Both N. pumila subsp. sinensis and N. japonica showed high H o levels of 1.0 and high H e levels of 0.7 and 0.8, respectively. In addition, locus AC182b was found to be monomorphic between the two species.

Table 3

Results of cross‐amplification of 39 newly developed microsatellite loci for Nuphar shimadai in N. pumila subsp. sinensis and N. japonica.a

Locus	Nuphar pumila subsp. sinensis (n = 5)				Nuphar japonica (n = 20)
	A	A e	H o	H e b	A	A e	H o	H e b
AG144	1	1.0	0.0	0.0	5	4.2	0.7	0.7ns
AG146	2	2.0	1.0	0.5***	7	4.3	0.9	0.8ns
AG152	2	2.0	1.0	0.5***	6	5.2	1.0	0.8***
AG158	2	1.5	0.0	0.3***	3	2.2	0.0	0.6***
AG159	1	1.0	0.0	0.0	3	2.8	0.8	0.7ns
AG164	2	2.0	1.0	0.5***	8	6.1	1.0	0.8***
AG167	2	1.5	0.0	0.3***	4	3.4	0.6	0.7ns
AC152	2	2.0	1.0	0.5***	3	2.4	0.3	0.6**
AG196	2	1.5	0.0	0.3***	4	3.1	0.1	0.7***
AG207	1	1.0	0.0	0.0	5	3.2	0.1	0.7***
AG223	2	2.0	1.0	0.5***	3	2.0	0.8	0.5**
AG224	2	2.0	1.0	0.5***	9	5.6	0.9	0.8ns
AG225	1	1.0	0.0	0.0	4	3.1	0.1	0.7***
AC139	1	1.0	0.0	0.0	4	3.2	0.8	0.7ns
AC143	1	1.0	0.0	0.0	7	4.8	0.1	0.8***
AC149	2	1.9	0.8	0.5ns	9	6.8	0.9	0.6**
AC150	2	1.5	0.4	0.3ns	7	4.0	0.5	0.8**
AC155	4	2.9	1.0	0.7***	8	4.2	0.5	0.7ns
AC165	1	1.0	0.0	0.0	5	2.4	0.0	0.6***
AC170	1	1.0	0.0	0.0	7	4.2	0.2	0.7***
AC171	2	1.2	0.2	0.2ns	7	3.8	0.3	0.7***
AC172	1	1.0	0.0	0.0	3	2.4	0.6	0.6ns
AC176	4	3.8	1.0	0.7***	5	2.3	0.1	0.6***
AC178	2	2.0	1.0	0.5***	5	3.5	0.3	0.7***
AC182a	1	1.0	0.0	0.0	3	2.2	0.0	0.6***
AC182b	1	1.0	0.0	0.0	1	1.0	0.0	0.0
AC186	2	2.0	1.0	0.5***	5	4.1	0.8	0.8ns
AC189	1	1.0	0.0	0.0	3	2.6	0.4	0.6ns
AC198b	1	1.0	0.0	0.0	7	3.7	0.2	0.7***
AC206	1	1.0	0.0	0.0	4	3.1	0.0	0.7***
AC136	1	1.0	0.0	0.0	2	2.0	0.4	0.5ns
AC111	2	1.9	0.0	0.5***	3	2.4	0.0	0.6***
AC112	2	2.0	1.0	0.5***	8	5.8	0.9	0.8ns
AC129	1	1.0	0.0	0.0	4	2.4	0.1	0.6***
AC130	2	1.9	0.0	0.5***	5	3.7	0.5	0.7ns
AC131	1	1.0	0.0	0.0	2	1.2	0.0	0.2***
AC132	1	1.0	0.0	0.0	6	3.6	0.3	0.7***
AG135	2	1.5	0.0	0.3***	4	3.6	0.0	0.7***
AC173	2	2.0	1.0	0.5***	9	4.1	0.5	0.8***
Mean	1.6	1.5	0.3	0.3	5.1	3.5	0.4	0.7

A = number of alleles; A e = number of effective alleles; H e = expected heterozygosity; H o = observed heterozygosity; n = number of individuals sampled.

aVoucher and locality information are provided in Appendix 1.

bDeviation from Hardy–Weinberg equilibrium: *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant.

CONCLUSIONS

In the present study, we developed 39 microsatellite loci in N. shimadai and demonstrated their cross‐amplification in two closely related species, N. pumila subsp. sinensis and N. japonica. For conservation purposes, these markers will be useful tools in the evaluation of genetic diversity of the endangered N. shimadai in Taiwan. Based on the applicability of these markers in the related species identified here, they will be useful in future investigations of genetic variation and evolutionary history of Nuphar and related taxa.

AUTHOR CONTRIBUTIONS

H.Y.L., H.C.S., L.P.J., and Y.C.C. conceived and designed the experiments. H.Y.L., H.C.S., L.P.J., C.C.H., and Y.C.C. performed the experiments. H.Y.L., H.C.S., and Y.C.C. analyzed the data. H.Y.L., L.P.J., C.C.H., and Y.C.C. contributed reagents, materials, or analysis tools. H.Y.L. and Y.C.C. wrote the paper. H.Y.L., H.C.S., L.P.J., C.C.H., and Y.C.C. conceived of the study, edited the manuscript, and approved the final manuscript.

DATA ACCESSIBILITY

Sequence information for the developed primers has been deposited to the National Center for Biotechnology Information (NCBI); GenBank accession numbers are provided in Table 1 and Appendix 2.

Species	Voucher specimen accession no.a	Collection locality	Locality code	Geographic coordinates	n
N. shimadai Hayata	Ju3171–Ju3192	Taoyuan, Taiwan	WP	24°53′16″N, 121°11′39″E	22
	Ju3206–Ju3216	Taoyuan, Taiwan	GPa	24°52′46″N, 121°11′33″E	11
	Ju3193–Ju3205	Taoyuan, Taiwan	GPb	24°52′44″N, 121°11′34″E	13
	Ju3230–Ju3245	Taoyuan, Taiwan	GPn	24°53′01″N, 121°11′36″E	16
	Ju2976–Ju3051; Ju3053–Ju3054	Ilan, Taiwan	LD	24°45′27″N, 121°35′38″E	78
N. pumila (Timm) DC. subsp. sinensis (Hand.‐Mazz.) Padgett	Ju3218; Ju3220	Jiangxi Province, China	GJ	29°33′09″N, 115°58′13″E	5
N. japonica DC.	Ju3221; Ju3224	Owase, Japan	JP	36°56′01″N, 139°17′42″E	20

Locus	Primer sequences (5′–3′)	Repeat motif	Allele size (bp)	GenBank accession no.
NS‐AG‐171	F: TACAAGGCATGTGTTACAGC	(TC)11	158	MH700494
	R: GAGACCTTACACTTGCCAAG
NS‐AG‐175	F: GAAGAAGTCTTACCTTGAAG	(GA)22	156	MH700495
	R: AAGATCAACCGGCTATCTTG
NS‐AG‐187	F: GGTCAATGAGAGTTTGTAGG	(TG)10	178	MH700496
	R: TATGTTTCCCTCGTGCACAG
NS‐AG‐216	F: AATGTCACCGTAGTTGTCAC	(AG)21	145	MH700497
	R: TACCCGTTGATAAGAGCGAC
NS‐AC‐145	F: AGGAAGATTGACCGAGAAGC	(AG)17	192	MH700498
	R: ATTGTTCGGTTCATGGGTTC
NS‐AC‐158	F: TTGCTCCTATATGACGGCTG	(AG)19	215	MH700499
	R: CAGCCGTCATATAGGAGCAA
NS‐AC‐168	F: TTTGGTTCATATCGCTGACC	(TTG)7	171	MH700500
	R: TCCCAAGGCTATTCCTTATG
NS‐AC‐175	F: ATCAGTCCCTATTGATCACG	(AG)23	152	MH700501
	R: ACGAGTTTTCTATTGTGCAG
NS‐AC‐196	F: ACCACCTCAACAATGGAGTC	(GA)20	157	MH700502
	R: AGAAAGTTATGATGGGGAGC
NS‐AC‐198a	F: CGACACCATGAGTCAATGAC	(TG)8	176	MH700503
	R: TGCCATGGTGTTGCCGAATG
NS‐AG‐58	F: ACTCACGTGCTGCGAACATG	(AG)28	195	MH700504
	R: GGGCAGAACGAGAAAATGCC
NS‐AG‐7	F: CGATGACATACATCCGTTGC	(TC)23	158	MH700505
	R: GATCATATTTGGAGCCCGAG
NS‐AG‐12	F: ACTAAGGGCATGTTTGGAAG	(AG)22	247	MH700506
	R: TGTTCTTGTTCAGTTCATGG
NS‐AG‐25	F: AGTGAGAAGACGTACTGAGG	(AG)16	162	MH700507
	R: GCTACAGGATTCTCAATGTTG
NS‐AG‐26	F: TTTGAACACCTCTCGGTAAC	(AG)40	203	MH700508
	R: GGAAGATTATTAGACCCTAG
NS‐AG‐37	F: CCAAACCTGGACAACAAAAC	(TC)9	124	MH700509
	R: AGCATCCAAGTCACTCAAGC
NS‐AG‐38	F: GTCTCAACCTTCTCCGAAGC	(AG)22	184	MH700510
	R: TAAGGTGGAAGAGGCAACCC
NS‐AG‐43	F: TAAGGCATGAAGGAAGAGAG	(TC)23	168	MH700511
	R: AACTAGTACTTGTATCCCTG
NS‐AG‐49	F: TGAACTTGTCATGCTGCACC	(AG)15	182	MH700512
	R: ACTAGTTTATGCATCCCACG
NS‐AC‐94	F: TAAGCAGGTAGATGCCTGTC	(TG)21	127	MH700513
	R: CAGCAAAGCACCAGTGCTAG
NS‐AC‐103	F: ATGAGTCGTGGGTAACCATG	(AC)9	149	MH700514
	R: GGGGGTCTTATGATAGCTTG
NS‐AC‐119	F: CCTAATAACAAGGGAGTTGG	(TC)20	171	MH700515
	R: ATATAGAAAGTGGGCCTGTG
NS‐AC‐120	F: TTGTTAGACTGAAAGAGTCG	(AG)9	141	MH700516
	R: CATTCATGCATGTCAATGTG
NS‐AC‐133	F: ATACTGCAACATACTCGAGC	(TG)9	160	MH700517
	R: TTTGGAGGAAAGGGATAAAC
NS‐AC‐134	F: ATGGATTCCACCATGAGTCC	(TC)20	171	MH700518
	R: TTCCCTCGTTGATCTTATTG
NS‐AG‐151	F: TGCACGTGAACTAAATCCTG	(AC)8	135	MH700519
	R: TTTTAGATCACAGCTGCATC
NS‐AG‐75	F: GATTGAGAAGATGCAACCTG	(AG)25	209	MH700520
	R: GATCCAAAGAATATGTTGCC
NS‐AG‐91	F: TTTTCACGGGTAGGATCTAC	(CT)25	226	MH700521
	R: ATTGACTGATACCCCTTACC
NS‐AG‐104	F: TTTTGCAACCATCTTTTGCC	(TC)24(AC)11	171	MH700522
	R: ATAGGTTGTCTACAATCCAG
NS‐AG‐119	F: CACGCTCCCAAGATCAATTG	(AG)18	158	MH700523
	R: TAACCCATGTATGGGTTGAG
NS‐AG‐153	F: TTCTAGCCAAAGCAAATAGG	(AG)9	164	MH700524
	R: GTCACGATTTCATTAGCAGC
NS‐AG‐65	F: TTTGATTCACACGGCCTGCC	(CT)8	121	MH700525
	R: TGCCTGCAGGTCGACTCTAG
NS‐AG‐113	F: TAACGATAACATGGGGAGTT	(CT)14	126	MH700526
	R: AAAATGAAGAGGGATGGTGG