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American Journal of Botany 91(12): 2022–2029. 2004.

CRYPTIC

SPECIES IN AN ENDANGERED PONDWEED

(POTAMOGETON, POTAMOGETONACEAE) REVEALED BY AFLP MARKERS1

COMMUNITY

JUSTEN B. WHITTALL,2,3 C. BARRE HELLQUIST,4 EDWARD L. SCHNEIDER,5 AND SCOTT A. HODGES3 Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, California 93106 USA; Department of Biology, Unit 9168, 375 Church Street, Massachusetts College of Liberal Arts, North Adams, Massachusetts 01247 USA; and 5Santa Barbara Botanic Garden, 1212 Mission Canyon Road, Santa Barbara, California 93105 USA 3

4

Cryptic species are morphologically indistinguishable, yet reproductively isolated. Morphological boundaries between species can also be obscured by hybridization and clonality. Determining the roles of reproductive isolation, hybridization, and clonality in morphologically indistinguishable taxa is essential to determining appropriate species-level taxonomic rankings for conservation purposes. The taxonomic status of the endangered Little Aguja pondweed of west Texas, Potamogeton clystocarpus, is uncertain due to a lack of fixed morphological differences between it and two sympatric congeners. Morphology, amplified fragment length polymorphisms (AFLPs), and sequences of the internal transcribed spacer (ITS) region and trnL-F intron and spacer were used to determine the degree of genetic distinctiveness, hybridization and clonality for this rare species. AFLPs indicate that P. clystocarpus is a genetically distinct lineage compared to P. pusillus and P. foliosus. No hybrids involving P. clystocarpus were detected, but two putative hybrids involving P. pusillus and P. foliosus were identified. Clonal growth was only detected in P. pusillus. A combination of morphological and molecular markers was successful in determining the genetic distinctiveness of an endangered cryptic species, Potamogeton clystocarpus. Further sampling in this and adjacent drainages is necessary to assess the degree of endemism of P. clystocarpus and confidently rule out hybridization and clonality in this taxon. Key words: ceae.

AFLP; aquatic plant; cryptic species; endangered species; hybridization; Potamogeton clystocarpus; Potamogetona-

During the process of speciation, two taxa become reproductively isolated, which leads to genetic and, usually, morphological divergence. Historically, fixed morphological differences have been used as a proxy for genetic divergence between taxa and the determination of species-level taxonomy. But, the absence of fixed morphological differences does not always indicate a lack of genetically distinct lineages. Cryptic species are morphologically indistinguishable, yet reproductively isolated (Grant, 1981; Paris et al., 1989). Determining appropriate species-level taxonomy for cryptic species requires a more direct estimate of reproductive isolation and genetic divergence. Molecular markers have provided an alternative estimate of genetic distinctiveness and a test for species-level status of many suspected cryptic plant species (Masuyama et al., 1994; Yatabe et al., 2001; Waycott et al., 2002). For example, molecular markers have identified genetically distinct serpentine forms of Lasthenia californica, which are morphologically indistinguishable (Rajakaruna et al., 2003). Cryptic species are particularly common in lineages that diversify in habitats that impose substantial physiological and morphological constraints. These constraints result in low or Manuscript received 25 February 2004; revision accepted 26 August 2004. The authors thank Dr. Douglas Bush, Ji Yang, and Brian Counterman for assistance in the laboratory and many helpful discussions. Dr. Robert Haynes generously provided an unpublished Potamogeton molecular phylogeny. John Karges provided access to The Nature Conservancy portions of Little Aguja and Madera Canyons. John Dee Johnson and Dan Damon provided access to the Buffalo Trail Boy Scout Ranch. Dr. Kathryn Kennedy, Center for Plant Conservation, provided early assistance on this project. This research was supported under cooperative agreement 1448-20181-00- J801 between the U.S. Fish and Wildlife Service, Region 2 and the Santa Barbara Botanic Garden and National Science Foundation DEB-9726272 to SAH. 2 E-mail: [email protected]. 1

non-existent morphological divergence between lineages, particularly in traits construed to be adaptive. Instead, often only small and seemingly trivial morphological differences arise (possibly through genetic drift of neutral characters) and are then used as a proxy for genetic distinctiveness and species delineation. Unfortunately, for taxonomic determination variation in such traits can also arise due to clonality and can be blurred by hybridization. Extensive clonality can generate large numbers of unique lineages, each with unique morphological characteristics (Clevering and Hundscheid, 1998). These fixed idiosyncrasies between clonal lineages can mimic the relatively small morphological divergences between cryptic species and therefore lead to taxonomic confusion. Hybridization, on the other hand, can generate individuals representing a morphological continuum between two species, thereby complicating traditional morphologically based taxonomies. Thus, taxonomic confusion can arise in cryptic taxa that hybridize, reproduce clonally, and occur in habitats that impose morphological constraints. Aquatic plant species are a prime example of lineages where taxonomic confusion is rampant, and the roles of clonality, hybridization, and cryptic species remain unresolved. The submerged environment restricts the potential morphology and physiology of many plant organs, thereby limiting the range of phenotypic differences between species (Niklas, 1997). In addition, aquatic plants are notorious for their propensity to reproduce vegetatively and hybridize (Les and Philbrick, 1993). Not surprisingly, therefore, substantial taxonomic confusion occurs in many aquatic genera (Haynes and Hellquist, 2000) and raises the question of whether cryptic species may actually underlie many of these subtle morphological variants. Identifying each of these factors is critical to understanding

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Fig. 1.

WHITTALL

ET AL.—AFLPS REVEAL CRYPTIC

POTAMOGETON

SPP.

2023

Location of the pools where collections were made from the Little Aguja Creek (A) and Madera Creek (M) in Jeff Davis County, west Texas, USA.

the evolutionary dynamics, appropriate taxonomy, and conservation status for rare species (Avise and Hamrick, 1996). The aquatic pondweed genus Potamogeton (Potamogetonaceae) has been cited for its prevalence of cryptic species, ability to hybridize extensively, and capacity for vegetative reproduction (Haynes and Williams, 1975; Hollingsworth et al., 1995, 1996; Mader et al., 1998; Fant et al., 2001a, b; Iida and Kadono, 2002). With over 100 species in the genus, between 50 and 200 unique taxonomic combinations have been suggested to be involved in hybridization (Les and Philbrick, 1993). Hybridization is facilitated by the high frequency of sympatric species due to their aquatic habitat requirements. In addition, the ability to reproduce vegetatively through the spread of over-wintering turions and rhizomes has been documented in many taxa (Ogden, 1966; Correll and Correll, 1972; Whittall et al., 2003). Thus, Potamogeton provides an ideal system to dissect the potential causes of taxonomic confusion: cryptic species, hybridization, and/or clonality. One species of Potamogeton, P. clystocarpus Fern., provides a particularly good example of the problems encountered in determining the taxonomic status of species in this genus. This species is considered one of the rarest plants in Texas, restricted to a single isolated source of perennial water in the sky-islands of the Davis Mountains, west Texas, and is listed as a federally endangered species due to its restricted distribution (U.S. Fish and Wildlife Service, 1990). Potamogeton clystocarpus co-occurs with four other Potamogeton species, two of which are nearly indistinguishable morphologically from P. clystocarpus; P. pusillus L. and P. foliosus Raf. (Whittall et al., 2003). Morphological identification of P. clystocarpus is especially difficult because the principal trait, the presence of basal tubercles on fruits, can only be observed once samples are dried (Fernald, 1932; Whittall et al., 2003). Due to this species’ aquatic habitat, and specifically, the tendency for the fruits to be submerged, dried fruits are rarely found in the field. Therefore, correct identification is nearly impossible unless fruits are collected and dried. Not surprisingly, the taxonomic distinctiveness of P. clystocarpus has been questioned

due to the deficiency of distinguishing morphological characteristics (Wiegleb and Kaplan, 1998). Molecular markers can provide insights into the genetic dynamics underlying morphologically indistinguishable taxa. Specifically, AFLP markers can differentiate closely related lineages, identify hybrids, and detect clonal growth as a genetic fingerprinting technique (Vos et al., 1995; Beismann et al., 1997; Aggarwal et al., 1999; O’Hanlon et al., 1999; Elias et al., 2000). We used AFLPs in combination with DNA sequence comparisons of the nuclear ribosomal ITS region (Baldwin et al., 1995) and the chloroplast trnL-F chloroplast intron and spacer (Taberlet et al., 1991) to test the hypothesis whether P. clystocarpus is a genetically unique cryptic species. Morphological identifications of dried material were conducted independently to substantiate the molecular data. The combined molecular and morphological data are interpreted in light of the current conservation status of P. clystocarpus in this endangered pondweed community. MATERIALS AND METHODS Sampling—Fresh leaf tissue was collected and dried in silica powder from 21 thin-leaved Potamogeton individuals from the Little Aguja Creek (A) and the adjacent Madera Creek (M) of the Davis Mountains, Jeff Davis County, west Texas (Fig. 1). An attempt was made to collect only P. clystocarpus leaves, but field identification is often inaccurate. Therefore, voucher specimens were made for each sample and deposited in the Massachusetts College of Liberal Arts Herbarium (NASC). Samples were collected to span the range of possible localities for P. clystocarpus. One to five individuals were collected at .1 m apart from five pools in Little Aguja Creek and three pools in Madera Creek (Table 1, Fig. 1). Samples were abbreviated using the creek designation (A or M), pool number, and individual number. For example, individual 1 from pool 2 of Little Aguja Creek is A2.1. Morphological analysis—Dried herbarium specimens were identified to species based on a suite of cryptic morphological characters (Whittall et al., 2003). These identifications were made concurrently with the molecular investigation, yet at a separate location.

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DNA extraction—DNA was extracted from 20 mg of dried leaf tissue following the protocol of the DNeasy Plant Mini Kit (Qiagen). Four DNA extractions were replicated to determine the repeatability of the AFLP technique.

TABLE 1.

AFLP analysis—The AFLP protocol followed a modified method of Vos et al. (1995). EcoRI and MseI were used in digestions of genomic DNA (250 ng). Sixteen selective primer pairs were screened using a subset of the samples to determine which primer pairs would provide the highest repeatability and scorable bands among replicated DNA extractions (M-CAA, M-CAT, MCAG, M-CAC, M-CTA, M-CTT, M-CTG, M-CTC with both E-AGC-700 and E-AAG-800 primers). Six of these primer pairs with the highest repeatability and number of scorable bands were used in selective amplifications of the 21 Potamogeton samples (M-CAA, M-CAT, and M-CAG with both E-AGC-700 and E-AAG-800). Products were denatured and separated on 7% acrylamide gels on a LI-COR 4200 DNA Sequencer. Polymorphic bands were scored manually. Genetic distances and neighbor joining phylogenetic analyses were performed with PAUP 4.0 beta 8 (Swofford, 2000). Bootstrap support for individual nodes was determined with 1000 replicates. Individuals displaying additive banding patterns from two distinct lineages were considered putative hybrids and removed from further AFLP analyses. Analysis of molecular variance was conducted in Arlequin v 2.000 (Excoffier et al., 1992) after removing the two P. foliosus samples because of their substantial divergences from the remaining samples.

P. P. P. P. P. P. P. P. P. P. P. P.

DNA sequencing—DNA sequencing of the trnL-F region and ITS region was performed on a subset of samples identified from morphology and AFLP analysis. These include P. clystocarpus (two samples), P. pusillus (three and four samples respectively), a New England collection of P. pusillus ssp. tenuissimus (Mertens and W. D. J. Koch) R. R. Haynes and Hellquist, the putative hybrid individuals (A1.4 and M3.4), and P. foliosus (two samples) (Table 1). Approximately 20 ng of template DNA were used in amplifications. The ITS region was amplified using primers ITS 5* (Liston et al., 1996) and ITS 26S– 25r (Nickrent et al., 1994). To amplify the trnL-F region, we used an M13 tailed primer ‘‘c’’ and primer ‘‘f’’ (Taberlet et al., 1991). Thermal-cycling conditions for ITS followed Whittall et al. (2000). Thermal-cycling conditions for the trnL-F region were 948C for 1 min followed by 35 cycles of 948C for 1 min, 558C for 45 s, 728C 45 s, finishing with 728C for 5 min. Amplification products were gel purified (Qiagen) and sequenced with a Thermosequenase Kit (Amersham) on a LI-COR 4200 DNA Sequencer with fluorescently-labeled ITS primers and fluorescently-labeled M13R primer for the trnL-F region. Contiguous sequences were assembled with Sequencher (Gene Codes) and aligned with GCG 10 (GCG, 1999). Maximum parsimony phylogenetic analyses of the DNA sequence data were conducted with PAUP 4.0 beta 8 (Swofford, 2000). ITS polymorphisms at variable sites were identified as superimposed nucleotide additivity patterns from chromatograms of direct sequences (Whittall et al., 2000). In addition, ITS indel polymorphisms were determined from the total sequence length and located at the site where there was a shift in the direct sequencing reading frame between the two ITS types (Whittall et al., 2000). RFLP analysis—Restriction sites (ScrFI for the ITS region and TaqI for the trnL-F intron and spacer) were identified that distinguished P. foliosus from the P. pusillus/P. clystocarpus lineage, which enabled us to quickly survey the remaining samples for their ITS and trnL-F types. Two to five units of restriction enzyme were combined with the appropriate 10% (w/v) buffer (New England Biolabs) and 500–750 ng of PCR product [1100% (w/ v) BSA for TaqI] in 25-mL reaction volumes. Digests were incubated for 4 h at 378C for ScrFI and 658C for TaqI, and then visualized on 1.5% agarose gels stained with ethidium bromide.

RESULTS Morphological identifications—In the field we attempted to identify and collect only P. clystocarpus samples using leaf width and length, peduncle shape and length, and fruit shape

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Potamogeton collection localities and sample sizes. Taxona

pusillus pusillus pusillus pusillus pusillus clystocarpus clystocarpus clystocarpus pusillus 3 foliosus pusillus 3 foliosus foliosus pusillus ssp. tenuissimus

Creek

Little Aguja Little Aguja Madera Madera Madera Little Aguja Little Aguja Little Aguja Little Aguja Madera Little Aguja New England

Pool no.

No. individuals

1 2 2 3 4 3 4 5 1 3 4 NA

4 3 1 2 1 4 1 1 1 1 2 1

a Species were identified by morphological analysis of dried material conducted independent of the molecular analysis.

and length as taxonomic characters (Whittall et al., 2003). Subsequently, when the samples had been dried, we were able to use the presence of the basal tubercles on the fruits as a final determining taxonomic character. Of the 21 collections, only six were ultimately identified as P. clystocarpus (Table 1). Thirteen were identified as P. pusillus and two as P. foliosus. Two of the samples identified as P. pusilllus, one from each drainage, exhibited intermediate morphology with P. foliosus and were recorded as putative hybrid plants (A1.4 and M3.4). AFLP markers—Genetic distinctiveness—The level of reproductive isolation separating cryptic species can be estimated from the number of unique bands fixed in each lineage (Wolfe et al., 1998). A total of 144 polymorphic AFLP bands were scored. Substantial genetic distance separated the three species independently identified by morphological analysis. The mean character difference between the two most similar lineages, P. pusillus and P. clystocarpus, was 43% (after removal of the two putative hybrid plants). There are 36 unique bands fixed in all P. pusillus samples that were absent in all P. clystocarpus samples. Furthermore, in phylogenetic analyses, these two lineages are supported by 100% and 94% bootstrap values for these two lineages, respectively (Fig. 2). An AMOVA analysis comparing the distribution of genetic variation within and between these two lineages indicates 77% of the genetic variance separates P. pusillus and P. clystocarpus. Although these two lineages can be found in the same drainage (Little Aguja), both species were never collected in the same pool. Potamogeton pusillus was restricted to the two uppermost pools, whereas P. clystocarpus was exclusively found in the three downstream pools. Interestingly, P. foliosus often grows in the same pools as both of these taxa (Whittall et al., 2003). There was also some genetic subdivision within the P. pusillus samples, such that all the samples from the upper Little Aguja pools are nearly monophyletic (except for A2.3) and the Madera samples from Pool 3 form a monophyletic group (Fig. 2). Hybridization—Hybrid individuals are identifiable by the additive banding patterns from both parents (Raamsdonk et al., 2000). The two putative hybrids identified with intermediate morphologies were found to have additive AFLP banding patterns. Specifically, samples A1.4 and M3.4 share all 36 of the unique bands defining the P. pusillus lineage, as well as 30

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POTAMOGETON

SPP.

2025

Fig. 2. Neighbor-joining analysis of 144 polymorphic AFLP bands with bootstrap values .70% indicated at the branches. Branches leading to unique species (based on independent morphological identifications) are labeled with arrows. Parents of putative hybrids are indicated with dotted lines.

and 36 of the 40 unique bands in the P. foliosus lineage, respectively (Table 2). After removing the additive P. foliosus bands from the putative hybrids, a phylogenetic analysis of the remaining bands indicated that the putative hybrids were collected in the same pool as their P. pusillus parent (Fig. 2). Specifically, putative hybrid sample A1.4 shares two unique bands with all other P. pusillus samples from Little Aguja Pool 1, and putative hybrid sample M3.4 shares two unique bands with the identical haplotypes M3.1/M3.3. Clonality and within-species variation—We tested the accuracy of using AFLPs to identify clones by replicated DNA extraction and AFLP fingerprinting from four different samples including P. foliosus, P. pusillus, and one putative hybrid. On average, the two fingerprints from each sample differed by

less than one band with a range of 0–2 differences of the 144 total bands scored. Thus, we considered two samples to be members of a clone if they differed by two or fewer bands. We found two pairs of identical haplotypes among the 21 samples. These samples were identified morphologically as P. pusillus: one pair from Little Aguja Pool 1 (A1.2/A1.3) and one pair from Little Aguja Pool 2 (A2.1/A2.2). Furthermore, the entire upper Little Aguja P. pusillus samples (except A2.3) differ by only two or fewer bands, indicating they are likely members of the same clone (Fig. 2). Clonality may also be present in the Madera drainage with only two bands differentiating samples M3.1 and M3.3. No P. clystocarpus clones were identified, but three pairwise comparisons of P. clystocarpus samples differ by only four bands (97.2% similarity) indicating very low within-species genetic variation compared

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TABLE 2. Two putative hybrids (P. pusillus 3 foliosus) are characterized by the fraction of each species’ unique AFLP bands, the presence of nucleotide additivity in the ITS region, and their chloroplast haplotype. Fraction of unique AFLP bands Putative hybrids

P. pusillus

P. foliosus

ITS additivity

A1.4 M3.4

33/33 33/33

30/40 36/40

NO YES

trnL-F haplotype

P. pusillus P. pusillus

to the genetic distance separating P. clystocarpus and P. pusillus. DNA sequencing—Nuclear ribosomal ITS region—We sequenced the nuclear ribosomal ITS region to determine whether variation in this region corroborated our AFLP findings (See Data Supplement accompanying the online version of this article). Eleven of the 13 variable sites (including indels) distinguished the P. foliosus samples from the remaining P. pusillus and P. clystocarpus samples (Table 3). The only site suggestive of the genetic distinctiveness of P. clystocarpus was a 2bp deletion (476–477). This site is polymorphic within each of three P. pusillus samples, suggesting a very close relationship between these two species (Table 3). The DNA sequences of the ITS region provide additional evidence that sample M3.4 is indeed a hybrid plant. Specifically, this sample was polymorphic for all eleven variable sites distinguishing P. pusillus and P. foliosus (Table 3). Alternatively, putative hybrid A1.4 was not polymorphic at any of these sites but, rather, matched the sequences found in the other P. pusillus samples. This result is consistent with the less complete AFLP band additivity for sample A1.4 (Table 2). All remaining samples were surveyed for their ITS type using restriction digestion at ScrFI that distinguishes P. foliosus from the P. pusillus/P. clystocarpus lineage. No additional samples contained the P. foliosus ITS type. Chloroplast trnL-F region—We sequenced the trnL-F intergenic spacer and intron in order to determine the lineage of the maternal parent in the two putative hybrids (See Data Supplement accompanying the online version of this article). Eight variable sites were found among the 10 samples sequenced representing all three species (Table 3). Seven variable sites separated P. foliosus from the P. pusillus/P. clystocarpus lineage. The remaining site is autapomorphic for the New England accession of P. pusillus ssp. tenuissimus. Both putative hybrids (A1.4 and M3.4) had trnL-F haplotypes identical to the P. pusillus samples. All remaining samples were surveyed with the restriction enzyme TaqI, which distinguished P. foliosus from P. pusillus/P. clystocarpus. All of these samples had the P. pusillus/P. clystocarpus banding pattern. DISCUSSION Rarely do the potential causes of morphological crypsis include reproductive isolation, hybridization, and clonality. Whereas reproductive isolation is predicted to lead to morphological distinctiveness, the latter two processes are more likely to blur taxonomic boundaries. The case of the endangered P. clystocarpus provides a testable taxonomic hypothesis of cryptic genetic distinctiveness amid the possibility of hybridization and clonality in a community of congeners.

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Morphological identifications—The taxonomic confusion surrounding P. clystocarpus was confirmed in our attempt to field identify 21 samples for this molecular study. In the field, pondweed experts correctly identified less than one in three samples. This statistic emphasizes the need for careful collection of mature fruiting material when making dried voucher specimens for P. clystocarpus. In addition, these results highlight the need for a molecular investigation into the degree of genetic distinctiveness for this endangered, yet controversial cryptic species. Genetic distinctiveness—The cryptic morphological characteristics that distinguish P. clystocarpus from its relatives in this pondweed community have led some to question the taxonomic distinctiveness of this species (Wiegleb and Kaplan, 1998). The AFLP data strongly supports the genetic distinctiveness of the P. clystocarpus lineage even in the presence of other closely related species. The correlation of a few cryptic morphological characters with a distinctive molecular fingerprint has also supported taxonomic distinctions in other close relatives of P. pusillus (Kaplan and Stepanek, 2003). The distinctiveness of the P. clystocarpus lineage is identified by 36 missing bands when compared to P. pusillus. Some of these bands are shared between both P. pusillus and P. foliosus, suggesting a loss of a substantial number of bands in P. clystocarpus. Such a large change in the genome suggests some cytological changes such as a loss of chromosome fragments. Aneuploid series are common in this genus and have been specifically identified in P. pusillus (2n 5 26 and 28) (Hollingsworth et al., 1998) suggesting possible cytological changes contributing to this AFLP pattern. Further cytological investigations are underway to examine this hypothesis (J. Whittall and D. Wilken, Santa Barbara Botanic Garden, unpublished data). Only one site in the ITS sequences partially corroborates the genetic distinctiveness of P. clystocarpus. This site is polymorphic in some P. pusillus individuals suggesting recent ancestry and possibly a progenitor-derivative relationship. Alternatively, this could be a unique site to P. clystocarpus followed by introgression into P. pusillus samples, but there is no AFLP evidence for interspecific hybridization involving P. clystocarpus and P. pusillus. Lack of substantial cpDNA divergence for sequences of the trnL-F intron and spacer for two morphologically distinctive Potamogeton spp. indicates that this is not a powerful marker for determining taxonomic boundaries for closely related Potamogeton spp., but may facilitate the identification of parentage in hybridizations between more phylogenetically distinct lineages (Fant et al., 2003). Hybridization—We used morphological intermediacy, AFLP band additivity, and ITS nucleotide additivity patterns to survey for hybridization in this endangered pondweed community. There was no evidence of hybridization involving P. clystocarpus. Alternatively, two hybrids were identified involving the other two taxa in this community. This is the first molecular evidence for hybrids between P. pusillus (2n 5 26 and 28) and P. foliosus (2n 5 26 and 28) (Hollingsworth et al., 1998). If cpDNA is maternally inherited (Birkey, 1995), then it appears P. pusillus was the maternal parent in both hybridization events involving P. foliosus. These two species can often be found growing intermingled within a pool where the inflorescences are held just above the water’s surface. In

clystocarpus clystocarpus pusillus pusillus pusillus pusillus ssp. ten. pusillus 3 foliosus pusillus 3 foliosus foliosus foliosus

P. P. P. P. P. P. P. P. P. P.

C C C C C C C C C/T T T

G G G G G G G G T T

159

C C C . . C C C C C C

54b

C C C C C C C C C/G G C/G

89

G G G G G G G G G/T T T

200

A A A A A T A A A A

347

A A A A A A A A A/. . .

209

T T T T T T T T G G

624


401


407

trnL-F

ITS

C C C C C C C C A A

681


467

C C C C C C C C A A

817

.. .. CT/.. CT/.. CT/.. CT CT CT/.. CT/.. CT CT

476–477

T T T T T T T T G G

845

A A A A A A A A A/T T A/T

503


547

A A A A A A A A G G

854

X X X X X X X X X/. . .

551–565c

POTAMOGETON

b

A A A A A A A A G G

232

C C C C C C C C A/C A A

102


A slash (/) indicates polymorphism. A period (.) represents a deletion. c The presence of a 15-bp insertion between bp551–565 is signified by an ‘‘X.’’

Little Aguja Little Aguja Madera Madera Madera New England Little Aguja Madera Little Aguja Little Aguja

Little Aguja Little Aguja Little Aguja Madera Madera Madera New England Little Ajuga Madera Little Aguja Little Aguja

14a

WHITTALL

a

clystocarpus clystocarpus pusillus pusillus pusillus pusillus pusillus ssp. ten. pusillus 3 foliosus pusillus 3 foliosus foliosus foliosus

P. P. P. P. P. P. P. P. P. P. P.

Locality

Variable sites in the ITS region and the trnL-F intron and spacer. The position (bp) for each variable site and the character state for each individual are indicated.

Species

TABLE 3.

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dense masses, wind, water, or insect pollination could have led to interspecific hybridization as in many other species of pondweeds (Les, 1998; King et al., 2002). The two putative hybrids have differing levels of incomplete molecular additivity, suggesting later generational hybrids or backcrosses. The most completely additive hybrid, sample M3.4, exhibits this pattern in both AFLP and ITS comparisons even though the paternal parental comparison is from the Little Aguja drainage (Table 2). Potamogeton foliosus was also common in Madera Creek (Whittall et al., 2003), but no samples from that location were included in this study. The paternal parent of putative hybrid M3.4 is probably from the Madera drainage since pollen transport between drainages is less likely. Although sterility of some hybrid pondweeds limits the potential for backcrossing and later generational hybrids (Hollingsworth et al., 1996), molecular evidence consistent with later generation hybrids is relatively common in Potamogeton (Fant et al., 2001b; Iida and Kadono, 2002) and is likely the case for the less additive putative hybrid, A1.4. Clonality—Extensive clonality can blur taxonomic boundaries and reduce genetic variation, both significant concerns in determining appropriate conservation status for endangered species. The ability of most linear-leaved Potamogeton spp. to produce overwintering turions, a form of vegetative reproduction, suggests that clonality may be a significant form of dispersal. A novel form of vegetative reproduction in Potamogeton, rhizomes, has been documented in all three species in this unique pondweed community (Whittall et al., 2003). Therefore, we sought to determine the extent of vegetative reproduction in this pondweed community. Although all six P. clystocarpus individuals were genetically distinct, given the small sample size, additional sampling would be necessary to convincingly reject its ability to reproduce clonally. Several clones were identified from a very limited sampling of P. pusillus individuals, yet these are unlikely to be the main cause of taxonomic confusion within P. clystocarpus. Overall, most species of Potamogeton have the ability to reproduce vegetatively, yet standing levels of clonality vary widely between populations, and taxa and overall seem to be less common than previously assumed (Les and Philbrick, 1993; Mader et al., 1998; King et al., 2002). Limited clonal establishment is consistent with the hydrologic cycles of the Davis Mountains where creek beds are scoured by intensive flooding followed by periods of very slow water movement when portions of the creek become completely dry. Accurate estimation of the degree of vegetative reproduction would require further sampling of the drainage. Therefore, these results provide a conservative estimate of the extent of clonality in this pondweed community. Conservation implications—The federally endangered P. clystocarpus is a genetically distinct cryptic species, not a hybrid species nor capable of forming extensive clonal populations. In the field it is nearly indistinguishable morphologically from P. pusillus and also confused with P. foliosus, even by experts familiar with the distinctive characters (Whittall et al., 2003). Although P. clystocarpus is a genetically distinct lineage based on AFLP makers, the lack of fixed ITS and/or cpDNA differences indicates it is a relatively recent lineage. The taxonomic confusion surrounding P. clystocarpus is not due directly to its involvement in hybridization with other pondweeds in this community, although hybrids between P.

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pusillus and P. foliosus do contribute to an additional degree of complexity in identifying species and hybrids in this community. Lastly, clonal growth was only identified in P. pusillus, but was unlikely the source of taxonomic confusion with P. clystocarpus. This study verifies that P. clystocarpus has the ability to reproduce by sexual means, yet intraspecific variation based on AFLP markers remains very low. The limited within-population genetic variation suggests a facultative selfing mating system. This mating system is consistent with the relatively low pollen/ovule ratios measured for several linear-leaved Potamogeton spp. including P. foliosus (Philbrick and Anderson, 1987). The limited amount of genetic variation within a creek suggests that additional populations from nearby drainages may harbor unique variation important to this species’ longterm survival. The current distribution of P. clystocarpus appears to be limited to a portion of the lower Little Aguja drainage (Fig. 1). The presence of the other congeners in other portions of this drainage and adjacent drainages including the Madera is suggestive that P. clystocarpus may be found elsewhere in the Davis Mountains. Our limited sampling from the upper portion of Little Aguja and the Madera drainage did not identify any P. clystocarpus samples. Additional sampling of the Madera and other neighboring drainages would be necessary to confidently determine the degree of endemism for this rare taxon. A combination of morphological and molecular approaches to identification may be necessary to discern this cryptic species in the diverse and endangered pondweed communities of the Davis Mountains in west Texas. LITERATURE CITED AGGARWAL, R. K., D. S. BRAR, S. NANDI, N. HUANG, AND G. S. KHUSH. 1999. Phylogenetic relationships among Oryza species revealed by AFLP markers. Genetics 98: 1320–1328. AVISE, J. C., AND J. L. HAMRICK [EDS.]. 1996. Conservation genetics: case histories from nature. Chapman and Hall, New York, New York, USA. BALDWIN, B. G., M. J. SANDERSON, J. M. PORTER, M. F. WOJCIECHOWSKI, C. S. CAMPBELL, AND M. J. DONOGHUE. 1995. The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Annals of the Missouri Botanical Garden 82: 247–277. BEISMANN, H., J. H. A. BARKER, A. KARP, AND T. SPECK. 1997. AFLP analysis sheds light on distribution of two Salix species and their hybrid along a natural gradient. Molecular Ecology 6: 989–993. BIRKEY, C. W. 1995. Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolution. Proceedings of the National Academy of Sciences, USA 92: 11331–11338. CLEVERING, O. A., AND M. P. J. HUNDSCHEID. 1998. Plastic and non-plastic variation in growth of newly established clones of Scirpus (Bolboschoenus) maritimus L. grown at different water depths. Aquatic Botany 62: 1– 17. CORRELL, D. S., AND H. B. CORRELL. 1972. Aquatic and wetland plants of southwestern United States. U.S. Environmental Protection Agency, Washington, D.C., USA. ELIAS, M., O. PANAUD, AND T. ROBERT. 2000. Assessment of genetic variability in a traditional cassava (Manihot esculenta Crantz) farming system, using AFLP markers. Heredity 85: 219–230. EXCOFFIER, L., P. SMOUSE, AND J. QUATTRO. 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131: 479– 491. FANT, J. B., E. M. KAMAU, AND C. D. PRESTON. 2003. Chloroplast evidence for the multiple origins of the hybrid Potamogeton 3 sudermanicusHagstr. Aquatic Botany 75: 351–356. FANT, J. B., C. D. PRESTON, AND J. A. BARRETT. 2001a. Isozyme evidence for the origin of Potamogeton 3 sudermanicus as a hybrid between P. acutifolius and P. berchtoldii. Aquatic Botany 71: 199–208.

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