Isozyme variation among populations of the clonal species, Phragmites australis (Cav.) Trin. ex Steudel

Aquatic Botany 63 (1999) 241±259

Isozyme variation among populations of the clonal species, Phragmites australis (Cav.) Trin. ex Steudel Dana Pellegrin1, Donald P. Hauber* Department of Biological Sciences, Loyola University, New Orleans, LA, 70118 USA Received 2 October 1998; accepted 23 November 1998

Abstract Few studies have examined population genetic variation in clonal, emergent, aquatic plant species. Phragmites australis (Cav.) Trin. ex Steudel is a clonal, cosmopolitan species common to marshes, estuarine, and other wetland habitats. With the exception of several European studies examining local variation, little is known about the distribution of genetic variation in this taxon, particularly in the U.S. In recent years, the rapid and invasive, vegetative spread of P. australis into disturbed marsh habitats in the U.S., particularly on the Eastern Seaboard and the Mississippi River delta, has sparked interest in its ecology and genetic structure. In this study, electrophoresis was used to analyze isozyme variation among 37 populations of P. australis from the eastern half of the U.S. The electrophoresis data strongly support a primarily vegetative mode of reproduction and spread. A total of 21 multilocus, isozymic phenotypes were identified among the 37 populations. All populations sampled along the Gulf Coast (GC) from Texas to the Florida panhandle (with the exception of the two populations from the Mississippi River delta) were uniform, sharing a single, multilocus phenotype. P. australis populations had lower levels of percent polymorphic loci and number of alleles per locus than typical asexual terrestrial species, but had a higher mean heterozygosity. Nei's genetic distance UPGMA depicts a substantial amount of geographic clustering of populations. However, populations described as `invasive' showed no genetic similarity to one another. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Phragmites australis; Poaceae; Common reed; Marsh reed; Clonal variation; Isozymes; Population genetics

* Corresponding author. Tel.: +1-504-865-2769; fax: +1-504-865-2920; e-mail: [email protected] 1 Present address: Intertek Testing Services Inc., Jefferson, LA 70123, USA. 0304-3770/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 3 7 7 0 ( 9 8 ) 0 0 1 2 0 - X

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1. Introduction The distribution of genetic variation within plant species and the genetic structure of plant populations are dependent on life history and other ecological factors (Loveless and Hamrick, 1984). Clonal species, according to Loveless and Hamrick, tend to have high levels of genetic divergence among populations and levels of genetic variation within populations that are dependent on whether the populations consist of a high number of genets (i.e. highly subdivided) or few genets (i.e. monomorphic). Ellstrand and Roose (1987) found the genetic structure of clonal plant populations to be typically multiclonal and consisting of intermediate levels of genotypic variation, though most of the species included in their survey were terrestrial. Les (1988) pointed out that clonality is quite extensive in aquatic species, and in a more recent survey of isozymic variation in aquatic species, Barrett et al. (1993) observed ``widespread monomorphism and weak population differentiation.'' Phragmites australis (Cav.) Trin. ex Steudel, the common or marsh reed, is an emergent aquatic grass that typically displays clonal growth. European studies of P. australis, examining genetic variation and population genetic structure, seem to support the summary on clonal variation by Loveless and Hamrick (1984) of high levels of genetic divergence among populations (KuÈhl and Neuhaus, 1993; Zeidler et al., 1994; Koppitz et al., 1997) and either homogeneous (Koppitz et al., 1997) or heterogeneous populations (McKee and Richards, 1996), or a combination of the two (KuÈhl and Neuhaus, 1993; Zeidler et al., 1994). P. australis is found on every continent except Antarctica (Tucker, 1990). In the United States, it has become the dominant plant species in many marshes and disturbed wetland habitats along the Eastern Seaboard, around the Great Lakes, and in the Mississippi River delta (Tucker, 1990; Hauber et al., 1991). In the Mississippi River delta, the marsh reed has displaced competing freshwater macrophytes (Hauber et al., 1991; Guerin and Hood, unpublished). This probably relates to its highly vegetative growth habit and to its ability to occupy habitats varying in salinity (Haslam, 1972). Recent invasions by P. australis of marshes along the Eastern Seaboard of the United States (Tucker, 1990; Marks et al., 1994) and in the Mississippi River delta (Hauber et al., 1991) demonstrate the need for an overall genetic assessment of the reed. Published studies of population genetic variation in P. australis have focused on geographically localized populations primarily in Europe (Djerbrouni, 1992; KuÈhl and Neuhaus, 1993; Neuhaus et al., 1993; Zeidler et al., 1994; McKee and Richards, 1996; Koppitz et al., 1997). Therefore, the objectives of the present study are to 1. survey isozymic variation among P. australis populations, primarily from the eastern half of the U.S. in order to determine patterns of distribution based on genetic structure, 2. compare levels of genetic diversity in P. australis with other clonal aquatic species, 3. determine if genetic similarities are present between aggressive phenotypes responsible for invasion of marshes along the Eastern Seaboard of the U.S. and the Mississippi River delta, and

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243

4. provide additional, baseline genetic data on levels of genetic variation within this cosmopolitan taxon. 2. Material studied and methods 2.1. Field sampling A total of 37 populations were surveyed from 12 states (Table 1). Twenty-one collections were made along the Gulf Coast from just north of Tivoli, TX, to Apalachicola in the Florida panhandle; 10 populations were sampled along the East Coast from just south of Titusville, FL, to New Bedford, MA; five were sampled in the Midwest from Illinois and Wisconsin; and one population was collected near Lajitas, TX, on the banks of the Rio Grande River (Fig. 1). One East Coast population from the Ft. Saybrook marshes in Connecticut (CT1) was included specifically because it was considered to consist of an aggressive, invasive phenotype (B. Lapin, personal communication). For similar reasons, a population from the Mississippi River delta (MSD1) that consisted of the dominant invasive biotype from that area (referred to as `background'; Hauber et al., 1991) was also included. Preliminary electrophoresis surveys examining 15 shoots/population over a 150±200 m range revealed populations of P. australis were generally uniclonal. These surveys included four populations along the Gulf Coast and several populations in the Mississippi River delta. As a result, electrophoresis sample sizes were reduced (n ˆ 4) and the number of populations sampled was increased in an effort to better evaluate the degree and geographic pattern of genetic variation. In each population, shoot tips were collected every 10 m along a transect. Specimens were placed immediately on ice and stored at 38C until tissue was processed. Unprocessed tissue was not stored for longer than four weeks. In all populations, standing infructescences (usually several months old) were collected (10 per population) when present and accessible, and examined for seed. 2.2. Enzyme electrophoresis Standard horizontal starch gel electrophoresis was performed on tissue cut from the inner leaf bases of the upper shoot tips and homogenized in cold 0.1 M phosphate grinding buffer (pH 7.5) containing 6% PVP (Soltis et al., 1983). The homogenate was stored on filter paper wicks or in microcentrifuge tubes at ÿ808C. The gel/electrode buffer selection and enzyme system assays were based on those of Hauber et al. (1991), with the following enzymes examined: aspartate aminotransferase (AAT), colorimetric esterase (EST), shikimate dehydrogenase (SKD), phosphoglucomutase (PGM), phosphoglucoisomerase (PGI), fructokinase (FRK), triose-phosphate isomerase (TPI), isocitrate dehydrogenase (IDH), and 6-phosphogluconate dehydrogenase (PGD). Gels were run at 25±30 mA for 12 h or until the marker dye had migrated 12 cm from the origin. Stained gels were photographed and bands recorded by illustration.

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Table 1 Collection codes and localities of sampled populations of Phragmites australis Collection code

State

County

Specific location

Coll. Date

GC

TX

Refugio

TX 35, 0.3 miles S of Guadalupe River bridge (N of Tivoli, TX); extensive for several miles. 28±300 N 96±530 W

4 Aug, '91

TX

Chambers

I-10, on E bank of Lost River W of Trinity River; abundant. 29±500 N 94±470 W

8 Aug, '91

TX

Jefferson

TX 82, S of MLK Memorial Bridge just S of Port Arthur; occasional. 29±510 N 93-570 W

24 Jul, '91

LA

Cameron

LA 82, W of junct. LA 27 (Holly Beach)  1mi; occasional. 29±460 N 93±290 W

24 Jul, '91

LA

Cameron

LA 82, W of Pecan Island 29 miles; extensive. 29±440 N 92-560 W

24 Jul, '91

LA

St. Mary

LA 83, W of US 90 18 miles, near railroad tracks along Exxon property; frequent. 29±470 N 91-460 W

24 Jul, '91

LA

St. Mary

US 90, W of Atchafalaya River bridge 1 mile. 29±420 N 91-140 W

24 Jul, '91

LA

Terrebonne

US 90, at St. Charles St. intersection in Houma, along canal; occasional-to-extensive for several miles W of this. 29±340 N 90±440 W

24 Jul, '91

LA

Plaquemines

LA 23, N of West Point-a-la-Hache 11.7 miles; occasional. 29±420 N 89±590 W

23 May, '91

LA

Plaquemines

LA 23, near supermarket in Venice; frequent. 29±160 N 89±210 W

23 May, '91

LA

St. Tammany

I-10, N side of Lake Pontchartrain bridge (W of Slidell); occasional; 2n ˆ 6x ˆ 72. 30±120 N 89±470 W

8 Jun, '91

MS

Harrison

US 90, at Veterans Ave. intersection in Biloxi; occasional. 30±220 N 88±510 W

8 Jun, '91

MS

Jackson

US 90, near Gautier Dental Center in Gautier. 30±200 N 88±380 W

8 Jun, '91

AL

Baldwin

I-10, along US 90/US 98 exit ramp on E bank of Mobile Bay. 30±410 N 88±000 W

8 Jun, '91

FL

Escambia

US 98, on E bank of Perdido Bay. 30±240 N 87±350 W

8 Jun, '91

FL

Santa Rosa

I-10, W bank of Pensacola Bay; small population. 30±330 N 87±110 W

9 Jun, '91

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Table 1 (Continued ) Collection code

State

County

Specific location

Coll. Date

FL

Okaloosa

US 98, near Howard Johnson Inn in Ft. Walton Beach. 30±230 N 86±370 W

8 Jun, '91

FL

Walton

FL 20, W of Portland 3 miles; small population; 2n ˆ 6x ˆ 72. 30±290 N 86±130 W

8 Jun, '91

FL

Franklin

US 98, W of Apalachicola 0.6 miles; extensive; 2n ˆ 6x ˆ 72. 29±430 N 85±000 W

8 Jun, '91

FL1

FL

Brevard

FL 528, W of I-95/FL 407 exit 0.5 miles; frequent. 28±240 N 80±510 W

30 May. '92

FL2

FL

Duval

FL 105, W of FL 9A 2.3 miles. along bank St. John's River; isolated population. 30±240 N 81±360 W

30 May '92

SC1

SC

Georgetown

US 17, S of North Santee River bridge 0.6 miles; occasional in marsh then extensive for several miles N. 33±120 N 79±240 W

31 May, '91

SC2

SC

Horry

US 17, S of SC 544 0.6 miles; occasional. 33±360 N 79±120 W

31 May, '91

NC

NC

Brunswick

NC 133, S of Town Creek bridge 3 miles; isolated population. 33±570 N 78±090 W

31 May, '91

VA1

VA

Northhampton General Booth Blvd. near Virginia Marine Science Museum in Virginia Beach. 36±550 N 76±010 W

15 Apr, '93

VA2

VA

Northhampton Holly Road and 28th Street in local residential park in Virginia Beach; large population. 36±550 N 76±000 W

17 Apr, '93

CT1

CT

Middlesex

Ft. Saybrook marsh S of I-95 and N of CT 154; large population (coll. by B. Lapin). 41±170 N 72±220 W

9 May, '93

CT 2

CT

LitchfieldCT

112 in Baver Woods near Lime Rock, calcareous wetlands (coll. by B. Lapin). 41±580 N 73-190 W

14 May, '93

MA

MA

Bristol

S of Fairhaven across bay from New Bedford (coll. by S. Darwin). 41±330 N 70±500 W

13 Jul, '92

IL1

IL

Clair

I-255, S of I-55/I-70 2 miles; small population, rare. 38±430 N 90±070 W

15 Jun, '92

IL2

IL

Grundy

I-55 at IL 47 overpass; occasional. 41±080 N 88±250 W

16 Jun, '92

WI1

WI

Fon du Lac

US 41, S of WI 145 0.5 miles;, small, isolated population. 17 Jun, '92 43±350 N 88±280 W

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Table 1 (Continued ) Collection code

State

County

Specific location

Coll. Date

WI2

WI

Waukesha

22 Jun, '92

WI3

WI

Brown

County Road G, S of County Road SS (Pewaukee) 1 mile; small, isolated population in cattail marsh. 43±040 N 88±140 W Irwin Ave. under I-43 overpass near Atkinson's marsh in Green Bay; extensive. 44±350 N 87±590 W

TX

TX

Brewster

Along banks of Rio Grande River S of Lajitas, Big Bend National Park; occasional (coll. by D.A. White). 29±120 N 103±380 W

4 Mar, '92

MSD1

LA

Plaquemines

Cadro Pass N of Gulf of Mexico 0.5 miles. in Garden Island Bay of Mississippi River delta. 29±040 N 89±110 W

12 May, '92

MSD2

LA

Plaquemines

Cadro Pass N of Gulf of Mexico 0.5 miles in Garden Island. Bay of Mississippi River delta, adjacent to MSD1. 29±040 N 89±110 W

12 May, '92

25 Jun, '92

2.3. Gel interpretation and genetic analysis The lack of sexual reproduction precluded a formal genetic analysis of P. australis. Also, the fact that some of our samples are tetraploid, some are hexaploid and some others of unknown ploidy makes it difficult to draw absolute genetic comparisons. There does not seem to be a precedent for analyzing populations of varying polyploid levels. We tried to be conservative in the number of isozymes identified. For instance, all populations sampled share the same, single Idh band although, most likely, there are two- or three-gene loci coding for an identical product. Nevertheless, we identified Idh conservatively as having a single isozyme. For the most part, if individuals shared a band, we presumed it represented the same allozyme. Genetic interpretation of banding patterns was based on known isozyme numbers in polyploid plants (Soltis and Soltis, 1989) and the active unit substructure of individual enzymes assayed (Weeden and Wendel, 1989). The presumed isozymes were numbered sequentially, beginning with the most anodal band(s), as were presumed allozymes, which were labeled alphabetically. The species level indexes of polymorphism were calculated according to Hamrick and Godt (1990) as were the overall mean population-level indexes of polymorphism, using version 3.3 of GeneStat-PC (Lewis and Whitkus, 1989). This included percentage of polymorphic loci (P), number of alleles per locus (A), and genetic diversity (He). The mean observed heterozygosity (HET) was calculated independently. GeneStat-PC was also used to calculate Nei's (1972) genetic identity (I) and distance (D). An average linker cluster analysis (UPGMA) was generated from the genetic distance matrix via SYSTAT version 5.2 (Wilkinson et al., 1992). F-statistics were not calculated on account of the lack of intrapopulational variation.

D. Pellegrin, D.P. Hauber / Aquatic Botany 63 (1999) 241±259 247

Fig. 1. Map of the eastern half of the continental United States showing locations of study populations of Phragmites australis.

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3. Results No seed was observed in any of the old infructescences examined in this study, with one exception where seeds were recovered from WI2. However, attempts to germinate were unsuccessful. Nine enzymes putatively coded by 17 loci were resolved by electrophoresis (Table 2). A grand total of 21 multilocus, isozymic phenotypes were identified among the 37 populations surveyed. For all populations, Aat-1, Idh, Pgd-1, and Pgd-2 were monomorphic. Nineteen populations sampled along the Gulf Coast from north of Tivoli, TX, to the Florida panhandle near Apalachicola were uniform, sharing a single, multilocus phenotype. For the sake of simplifying the analysis, these 19 populations were, in most cases, designated collectively as `GC' (Gulf Coast). Otherwise, each population, with the exceptions of SC1 and SC2, was uniform and possessed a single, unique multilocus phenotype. SC1 and SC2 each contained two unique phenotypes. The two populations from the Mississippi River delta (MSD1 and MSD2), although from the same general geographic region on the Gulf Coast, did not share the GC phenotype. At the species level, P. australis is polymorphic at 41.2% of its loci (Ps), with 1.47 alleles per locus (As), and a genetic diversity (Hes) of 0.4218. The mean polymorphic indexes for population-level variation, Pp, Ap, Hep, and HET (ˆobserved heterozygosity) are 24.98%, 1.24, 0.1349, and 0.2476, respectively. The mean identity based on Nei's genetic identity values (I) was 0.722. These values ranged from 1.000 (between the population pairs FL1 and FL2, and between FL1 and GC) to 0.2782 (between MSD1 and IL1; Table 3). The UPGMA dendogram, using Nei's genetic distance as the scale (Fig. 2) depicts a substantial amount of geographic clustering of populations. Populations from the extreme southern United States (FL1, FL2, TX and GC), with the exception of the Mississippi River delta, display a relatively high degree of similarity and are strongly differentiated from all the other populations analyzed. The Mississippi River delta populations (MSD1 and MSD2) are also very similar, yet show little or no similarity to the other southern populations. Populations along the East Coast, from South Carolina (SC1) to Massachusetts (MA), form a distinct cluster, although considerable variability among the populations is apparent. The five Midwest populations (with the exception of IL1, the southernmost Midwest population) share a greater degree of genetic similarity than with any of the other populations sampled. Also apparent from the UPGMA is the lack of similarity between the described `invasive' phenotypes, CT1 and MSD1, and the relative similarity of CT1 to the other East Coast populations, at least some of which are considered to be stable and noninvasive (B. Lapin, personal communication; Hauber and Pellegrin, personal observation). Tetraploid chromosome counts of 2n ˆ 4x ˆ 48 were previously obtained for MSD1 and MSD2 (Gaudreault et al., 1989) and, more recently, three GC sites (St. Tammany Parish, LA; Walton County, FL; and Franklin County, FL) were determined to be hexaploid (2n ˆ 6x ˆ 72). Otherwise, no additional chromosome counts for other populations were obtained.

Table 2 Allele frequencies for populations of Phragmites australis using collection codes given in Table 1. Nineteen Gulf Coast populations are coded collectively as `GC' Locus Aat-1 Aat-2

Aat-4 Est

Skd-1

Skd-2

Pgm

FL2

SC1

SC2

NC

VA1 a VA2 a CT1 a CT2 a MA

IL1

IL2

WI1

WI2

WI3

TX

MSD1 MSD2

1.00 Ð 1.00 Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð Ð 0.50 Ð 0.50 1.00 Ð Ð Ð Ð Ð 0.50 0.50 Ð Ð Ð nd nd nd nd

1.00 Ð 1.00 Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð Ð 0.50 Ð 0.50 1.00 Ð Ð Ð Ð Ð 0.50 0.50 Ð Ð Ð Ð 0.50 Ð 0.50

1.00 Ð 1.00 Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð Ð 0.50 Ð 0.50 1.00 Ð Ð Ð Ð Ð 0.50 0.50 Ð Ð Ð Ð 0.50 Ð 0.50

1.00 Ð 1.00 Ð Ð 1.00 Ð Ð Ð Ð 1.00 Ð 1.00 Ð Ð Ð 0.25 0.25 Ð 0.50 Ð Ð Ð Ð 0.75 0.25 Ð Ð 0.50 Ð 0.50

1.00 Ð 1.00 Ð Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð 1.00 Ð Ð 0.25 0.25 Ð 0.50 Ð Ð Ð Ð 0.75 0.25 Ð Ð Ð 1.00 Ð

1.00 Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð 1.00 Ð 1.00 Ð Ð Ð 0.50 Ð Ð 0.50 Ð Ð Ð 0.50 Ð 0.50 Ð Ð Ð 1.00 Ð

nd nd nd nd nd nd nd nd nd nd nd nd 1.00 Ð Ð Ð Ð Ð Ð 1.00 Ð Ð Ð Ð 0.50 0.50 Ð nd nd nd nd

1.00 Ð 1.00 Ð Ð Ð Ð Ð 1.00 Ð 1.00 Ð Ð 0.50 Ð 0.50 1.00 Ð Ð Ð Ð Ð 0.50 0.50 Ð Ð Ð Ð 0.50 Ð 0.50

1.00 Ð 1.00 Ð Ð Ð Ð Ð 1.00 Ð 1.00 Ð Ð Ð 1.00 Ð 1.00 Ð Ð Ð Ð Ð 0.50 0.50 Ð Ð Ð 1.00 Ð Ð Ð

1.00 Ð 1.00 Ð Ð Ð Ð Ð 1.00 Ð 1.00 Ð Ð Ð 1.00 Ð 1.00 Ð Ð Ð Ð Ð 0.50 0.50 Ð Ð Ð Ð Ð Ð 1.00

1.00 Ð 1.00 Ð Ð Ð Ð Ð Ð Ð 1.00 Ð Ð Ð 1.00 Ð 1.00 Ð Ð Ð Ð Ð 0.50 0.50 Ð Ð Ð Ð Ð 1.00 Ð

1.00 1.00 Ð 1.00 Ð Ð Ð Ð Ð Ð 1.00 Ð Ð Ð 1.00 Ð 1.00 Ð Ð Ð Ð Ð 0.50 0.50 Ð Ð Ð Ð Ð Ð Ð

1.00 Ð 1.00 Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð Ð 0.50 Ð 0.50 1.00 Ð Ð Ð Ð Ð 0.50 0.50 Ð Ð Ð 0.50 Ð Ð 0.50

1.00 1.00 Ð Ð Ð Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð 1.00 Ð Ð 0.50 0.50 Ð Ð Ð Ð Ð 1.00 Ð Ð Ð Ð 1.00 Ð

nd nd nd nd nd nd nd nd nd nd nd nd Ð 1.00 Ð Ð 0.50 Ð Ð Ð Ð 0.50 Ð Ð 0.50 0.50 Ð nd nd nd nd

nd nd nd nd nd nd nd nd nd nd nd nd Ð 1.00 Ð Ð 0.50 Ð Ð Ð Ð 0.50 Ð Ð 1.00 Ð Ð nd nd nd nd

nd nd nd nd nd nd nd nd nd nd nd nd 1.00 Ð Ð Ð 0.50 Ð Ð Ð Ð 0.50 Ð Ð 1.00 Ð Ð nd nd nd nd

1.00 Ð 1.00 Ð Ð Ð Ð Ð 1.00 Ð 1.00 Ð 1.00 Ð Ð Ð Ð Ð Ð 0.50 0.50 Ð Ð Ð 0.50 Ð 0.50 Ð 0.50 Ð 0.50

1.00 1.00 Ð Ð Ð Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð Ð 1.00 0.50 Ð Ð 0.50 Ð Ð Ð Ð 1.00 Ð Ð Ð Ð 1.00 Ð

249

FL1

D. Pellegrin, D.P. Hauber / Aquatic Botany 63 (1999) 241±259

Aat-3

a a b a b c d e f a b c a b c d a b c d e f a b c d e a b c d

GC

250

Table 2 (Continued ) GC

FL1

FL2

SC1

SC2

NC

VA1 a VA2 a CT1 a CT2 a MA

IL1

IL2

WI1

WI2

WI3

TX

MSD1 MSD2

e a b c d Frk-2 a b c d Tpi-1 a b c Tpi-2 a b c d e Pgi-1 a b c d Pgi-2 a b c d Idh a Pgd-1 a Pgd-2 a

nd Ð Ð 0.50 0.50 Ð Ð Ð 1.00 Ð 1.00 Ð Ð 0.50 Ð 0.50 Ð 0.50 Ð 0.50 Ð Ð Ð Ð 1.00 1.00 1.00 1.00

Ð 0.50 Ð Ð 0.50 Ð Ð Ð 1.00 Ð 1.00 Ð Ð 0.50 Ð 0.50 Ð 0.50 Ð 0.50 Ð Ð Ð Ð 1.00 1.00 1.00 1.00

Ð 0.50 Ð Ð 0.50 Ð Ð Ð 1.00 Ð 1.00 Ð Ð Ð Ð 1.00 Ð 0.50 Ð 0.50 Ð Ð Ð Ð 1.00 1.00 1.00 1.00

Ð nd nd nd nd nd nd nd nd Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð Ð 1.00 Ð Ð Ð 1.00 Ð 1.00 1.00 1.00

Ð nd nd nd nd nd nd nd nd Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð Ð 1.00 Ð Ð Ð 1.00 Ð 1.00 1.00 1.00

Ð 0.50 Ð Ð 0.50 Ð Ð Ð 1.00 Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð Ð 1.00 Ð Ð Ð 1.00 Ð 1.00 1.00 1.00

nd nd nd nd nd nd nd nd nd nd nd nd Ð Ð Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð 1.00 Ð 1.00 1.00 1.00

Ð 0.50 Ð Ð 0.50 Ð Ð 1.00 Ð Ð 1.00 Ð Ð Ð 1.00 Ð Ð Ð Ð Ð 1.00 Ð Ð 1.00 Ð 1.00 1.00 1.00

Ð 0.50 Ð Ð 0.50 1.00 Ð Ð Ð Ð Ð 1.00 Ð Ð Ð 1.00 Ð Ð Ð 0.50 0.50 Ð Ð 1.00 Ð 1.00 1.00 1.00

Ð Ð Ð Ð 1.00 Ð 1.00 Ð Ð Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð 1.00 Ð 1.00 1.00 1.00

Ð 0.50 Ð Ð 0.50 1.00 Ð Ð Ð 1.00 Ð Ð Ð Ð Ð 0.50 0.50 Ð Ð Ð 1.00 Ð Ð 1.00 Ð 1.00 1.00 1.00

1.00 nd nd nd nd nd nd nd nd Ð 1.00 Ð 0.50 Ð Ð 0.50 Ð Ð Ð Ð 1.00 Ð Ð 1.00 Ð 1.00 1.00 1.00

Ð 0.50 Ð Ð 0.50 Ð Ð 1.00 Ð Ð 1.00 Ð Ð 0.50 Ð 0.50 Ð 0.50 Ð 0.50 Ð Ð Ð Ð 1.00 1.00 1.00 1.00

Ð Ð 0.50 Ð 0.50 Ð Ð Ð 1.00 1.00 Ð Ð Ð Ð Ð 1.00 Ð Ð 1.00 Ð Ð 1.00 Ð Ð Ð 1.00 1.00 1.00

Frk-1

a

nd ˆ no data or unresolved.

nd nd nd nd nd nd nd nd nd nd nd nd Ð Ð Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð 1.00 Ð 1.00 1.00 1.00

nd nd nd nd nd nd nd nd nd nd nd nd Ð Ð Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð 1.00 Ð 1.00 1.00 1.00

nd nd nd nd nd nd nd nd nd nd nd nd Ð Ð Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð 1.00 Ð 1.00 1.00 1.00

Ð 0.50 Ð Ð 0.50 Ð Ð Ð 1.00 Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð Ð Ð 1.00 Ð Ð 1.00 Ð 1.00 1.00 1.00

Ð 0.50 Ð Ð 0.50 Ð Ð Ð 1.00 1.00 Ð Ð Ð Ð Ð 1.00 Ð Ð 1.00 Ð Ð Ð 1.00 Ð Ð 1.00 1.00 1.00

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Locus

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Table 3 Nei's genetic identities (above) and distances (below) for pairwise comparison of populations of Phragmites australis using collection codes given in Table 1 GC GC FL1 FL2 TX SC1 SC2 NC MSD1 MSD2 MA IL1 IL2 WI1 WI2 WI3 VA1 VA2 CT1 CT2

GC FL1 FL2 TX SC1 SC2 NC MSD1 MSD2 MA IL1 IL2 WI1 WI2 WI3 VA1 VA2 CT1 CT2

0.0000 0.0132 0.0704 0.7116 0.6282 0.4775 0.8046 0.6562 0.6663 0.5074 0.7433 0.6027 0.6300 0.5978 0.6393 0.4788 0.5139 0.6316

FL1

FL2

TX

SC1

SC2

NC

MSD1

MSD2

MA

IL1

1.0000

0.9869 1.0000

0.9321 0.9395 0.9203

0.4909 0.5147 0.5432 0.4941

0.5335 0.5030 0.5309 0.5030 0.8853

0.6203 0.6075 0.6291 0.5381 0.8095 0.7911

0.4473 0.4260 0.4507 0.3579 0.4438 0.5076 0.4657

0.5188 0.5113 0.5342 0.4431 0.4627 0.5261 0.5138 0.8356

0.5136 0.5526 0.5774 0.4605 0.9162 0.7746 0.8158 0.4601 0.4942

0.6021 0.6369 0.6239 0.6900 0.6889 0.6732 0.6835 0.2782 0.3601 0.7076

0.0000 0.0624 0.6642 0.6872 0.4984 0.8532 0.6709 0.5931 0.4512 0.7568 0.5880 0.6476 0.6532 0.6393 0.4788 0.5139 0.6316

0.0831 0.6102 0.6332 0.4634 0.7969 0.6270 0.5491 0.4718 0.7033 0.5480 0.6343 0.6399 0.5697 0.4142 0.4493 0.5547

0.7050 0.6872 0.6197 1.0276 0.8140 0.7754 0.3711 0.6827 0.5880 0.6476 0.6532 0.6393 0.4788 0.5139 0.6316

0.1218 0.2114 0.8123 0.7706 0.0875 0.3727 0.4720 0.2644 0.4720 0.3937 0.0153 0.1706 0.1686 0.0328

0.2344 0.6781 0.6424 0.2554 0.3957 0.4950 0.3401 0.3737 0.4167 0.1330 0.0300 0.0328 0.1686

0.7643 0.6658 0.2036 0.3806 0.5190 0.3996 0.3132 0.4042 0.0434 0.1896 0.2639 0.1155

0.1796 0.7763 1.2793 0.8738 0.8735 0.6587 0.7772 0.7397 0.5924 0.5222 0.5222

IL2

WI1

WI2

WI3

VA1

VA2

CT1

CT2

0.4755 0.4692 0.4949 0.5053 0.6237 0.6096 0.5951 0.4173 0.4007 0.6496 0.6585

0.5474 0.5554 0.5781 0.5554 0.7676 0.7117 0.6706 0.4175 0.3854 0.7637 0.7668 0.7821

0.5326 0.5233 0.5303 0.5233 0.6237 0.6882 0.7311 0.5175 0.5008 0.5774 0.6932 0.7778 0.7141

0.5500 0.5204 0.5274 0.5204 0.6745 0.6592 0.6675 0.4597 0.4243 0.6553 0.7002 0.7157 0.8107 0.7346

0.5276 0.5276 0.5657 0.5276 0.9849 0.8754 0.9575 0.4773 0.5303 0.9575 0.6566 0.6019 0.7424 0.6019 0.7113

0.6196 0.6196 0.6608 0.6196 0.8432 0.9705 0.8273 0.5530 0.5837 0.7955 0.7636 0.7636 0.7987 0.7636 0.7636 0.7987

0.5982 0.5982 0.6381 0.5982 0.8448 0.9677 0.7680 0.5932 0.6229 0.7987 0.7373 0.7373 0.7712 0.7373 0.7373 0.7712 0.9831

0.5317 0.5317 0.5742 0.5317 0.9677 0.8448 0.8909 0.5932 0.6229 0.9216 0.6759 0.7373 0.7712 0.7373 0.7373 0.8898 0.8602 0.8898

0.2457 0.2513 0.3345 0.5077 0.2697 0.3048 0.3048

0.3367 0.2098 0.2978 0.2247 0.2598 0.2598

0.3085 0.5077 0.2697 0.3048 0.3048

0.3407 0.2697 0.3048 0.3048

0.2247 0.2598 0.1167

0.0171 0.1506

0.1167

0.7048 1.0215 0.9147 0.9535 0.6915 0.8572 0.6343 0.5384 0.4734 0.4734

0.3458 0.4314 0.2695 0.5491 0.4227 0.0434 0.2288 0.2247 0.0816

0.4177 0.2655 0.3664 0.3565 0.4207 0.2697 0.3048 0.3918

4. Discussion 4.1. Evidence for clonal propagation These results support the clonal nature of P. australis with its growth and spread primarily due to vegetative propagation. This mode of reproduction would

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Fig. 2. UPGMA dendogram with scale based on Nei's genetic distance for Phragmites australis populations using collection codes given in Table 1.

account for the apparent uniclonal nature of the majority of P. australis populations surveyed in this study where all but two populations, apparently, consist of single clones based on isozymes. More supportive is the discovery that 19 populations stretching virtually over the entire Gulf Coast appear to share the same genet (i.e. GC). The failure to detect any seed in most of the P. australis populations sampled may lie in the fact that the infructescences were relatively old, 6±8 months in most instances. However, MSD1 and MSD2 were carefully examined for seed set in one study (Fournier et al., 1995) and local populations of GC have been examined over several years, one and two months following flowering, but no seed has been recovered (personal observation).

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Table 4 Comparison of mean population-level measures of genetic diversity between Phragmites australis, two other aquatic species, and summary data for sexual/asexual (mostly terrestrial) species Taxon/grouping

Ap

PP

Hep

P. australis Vallisneria americana a Zostera marinaa Sexual/asexual (56 taxa) b

1.24 1.40 1.30 1.47

0.250 0.385 0.1834 0.294

0.135 0.085 0.063 0.103

a b

From Laushman, 1993. From Hamrick and Godt, 1990.

4.2. Population genetic structure Considering that the population-level diversity analysis in this study is based on small population sample sizes (n ˆ 4), we are hesitant to draw any broad conclusions regarding the significance of the measures of diversity (Pp, Ap, Hep, HET, I, and D) and the number and distribution of multilocus phenotypes. Nevertheless, some comparisons to other clonal species are warranted, particularly since such comparisons have not been made in past studies relating to Phragmites. Table 4 allows a comparison of our results to a summary of 56 sexual and asexual mostly terrestrial species (Hamrick and Godt, 1990), and to two other clonal aquatic species (Laushman, 1993). The means of two of the measures of diversity (Ap and Pp) were higher for the 56 terrestrial species (1.47 and 29.4%) than for P. australis (1.24 and 25%). These same measures for the four aquatic species were, on average, similar (with P slightly lower) to P. australis. Laushman (1993), in his focus on population genetics of clonally reproducing aquatic angiosperms, points out that aquatic species (in contrast to terrestrial species) have a greater ability to spread more rapidly by fragmentation leading to lower levels of genetic variation and lower levels of sexual reproduction for facultative asexual species. The mean heterozygosity (Hep) is indeed lower for the four aquatic species (0.062) in his survey when compared to the sexual and asexual terrestrial species (0.103), but P. australis had a Hep (0.1349) higher than either of these. This may be a function of the fixed heterozygosity (typical of many allopolyploids) of the individual clones that occupy each P. australis population. In contrast to this elevated heterozygosity, the number of genotypes per population in P. australis (0.57) is much lower in comparison to Zostera and Vallisneria (Laushman, 1993). The lower frequency may be an artifact of our very small sample size (n ˆ 4). Nevertheless, geographically localized populations of Zostera marina had 6.0 multilocus genotypes per population (all populations from tidal pools in False Bay, San Juan Island; Laushman, 1993); and, in a sample of 192 Vallisneria americana plants from a single population, 21 multilocus genotypes were identified (Laushman, 1993). However, given that Vallisneria and Zostera have a greater number of multilocus genotypes, and that some degree of sexual propagation can and does occur in Vallisneria and Zostera, one might expect the other measures of genetic diversity (i.e., Pp, Ap, and Hep) to be higher in Vallisneria and Zostera. Philbrick and Les (1996) suggest that the stability of the aquatic

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environment for submerged species, such as Vallisneria and Zostera, particularly over evolutionary time frames may help explain their low measures of genetic diversity as opposed to the slightly less stable environments of emergent species like P. australis. The lack of genetic variation among the Gulf Coast populations indicates that sexual propagation in P. australis, if and when it occurs, is largely inconsequential in this region. The same may not be true for the upper Midwest and East Coast populations. Seed set was reported as `abundant' in populations in Minnesota (Harris and Marshall, 1960). Seed set has also been observed in salt marsh populations from the Delaware Bay (Wijte and Gallagher, 1996), in isolated populations in Massachusetts and Connecticut (Hauber, 1990, personal observation), and in the WI2 population (Pewaukee, WI) in this study. In a study of populations in Quebec looking at seed production and chromosome number, 64 of the 73 populations sampled had at least one individual with seeds (Gervais et al., 1993). McKee and Richards (1996) concluded that climatic factors are important for seed set in Phragmites. In particular, a rainy pollination period seems to have a negative impact. Therefore, the genetic uniformity in the U.S. Gulf Coast populations might be attributed to climatic differences impacting seed set although the specific conditions are not clear. Other studies of P. australis have also revealed high levels of among population genetic variation. Zeidler et al. (1994) performed a molecular study based on three RFLP probes using four different restriction enzymes revealing 15±26 highly polymorphic bands. Sixteen populations were sampled in a relatively small geographic area along the shoreline of Lake Ammersee (15  5 km2) just southwest of Munich, Germany, with small sample sizes (n ˆ 2±4); 12 of these populations each consisted of a single genet. A total of eight genets were shared among the 12 clonal populations. Yet the eight genets were highly differentiated genetically. The fact that these genets occurred in a relatively small area suggests sexual propagation as seed production occurs in European populations (Haslam, 1972). In a somewhat broader isozyme study involving eight populations from western France and northern Algeria (sample sizes: n ˆ 3), each population was uniclonal (based on the zymograms of six enzymes), possessing a unique, multi-zymogram phenotype (Djerbrouni, 1992). Again, high variability was observed among genets. Finally, a recent study involving eight amply sampled populations from Germany, Hungary and Denmark using PCR fingerprinting revealed high differentiation among the populations (Koppitz et al., 1997). No genets were shared between populations, and four populations were uniclonal. Although typical measures of genetic diversity were not used in these European studies, and although PCR fingerprinting techniques generally reveal higher levels of genetic diversity than isozymes (Okoli et al., 1997; Sonnante et al., 1997) the results are consistent with the high variability reported in our study, particularly with respect to East Coast and Midwest populations. 4.3. Patterns of distribution and divergence The most recent comprehensive review reporting on mean genetic identities among conspecific plant populations gives an average identity of 0.95 out of 38 taxa and a range of 0.80±1.00 (Crawford, 1983). Nei's mean genetic identity for the P. australis populations analyzed is 0.722 (when each of the 19 Gulf Coast populations is considered

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individually rather than collectively), which is below the range of most conspecific plant populations. Other reports of taxa with similarly low conspecific mean identities (reviewed by Crawford, 1990; Chap. 7) include Bidens discoidea (0.688) and Hemeonitis (0.78). Les (1991) reported a low mean identity of 0.69 for Ceratophyllum echinatum. Ceratophyllum, unlike Bidens or Hemeonitis, resembles P. australis in that it is clonal and aquatic. Most gene flow in clonal aquatic species is attributed to vegetative fragmentation rather than from pollen or seed (Philbrick and Les, 1996). Clearly, Philbrick and Les (1996) were primarily referring to submerged aquatics. Although Europeon populations of Phragmites appear to employ seed dispersal over long distances since clones are not found to occur in more than one lake or population (Djerbrouni, 1992; Zeidler et al., 1994; Koppitz et al., 1997), nevertheless, P. australis plant fragments may have been dispersed over long distances (perhaps due to human influences) contributing to a low gene flow and high differentiation among U.S. P. australis populations. Also, since P. australis is a cosmopolitan taxon, the potential exotic origin of at least one population cluster (MSD1/MSD2 ± see below) in this study would be expected to lead to a lower overall genetic identity. The high degree of genetic differentiation suggests that undertaking a study combining isozymes, morphology, life history and other characters would be needed to better illuminate infraspecific relationships in P. australis. 4.4. Patterns of geographic differentiation This study provides strong evidence of the correspondence between certain geographic regions in the U.S. and the isozymic similarity among populations of P. australis in those regions, and this is most apparent in the UPGMA (Fig. 2). The most obvious is the Gulf Coast cluster including GC, FL1, FL2, and TX. The fact that the GC cluster is so extensive when considering the variants from Lajitas, TX (TX) and eastern Florida (FL1 and FL2), and yet does not appear particularly aggressive in the populations where it was collected (personal observation), suggests that the vegetative spread and colonization has been relatively gradual and that the phenotype probably represents a native type. Even so, perhaps the more interesting question is, what is the nature of the ecological tolerances of the GC phenotype and its variants from the desert climate and moderate elevation (640 m) along the banks of the Rio Grande River in southwest Texas (TX) to the subtropical freshwater, lowland marsh of eastern Florida (FL1)? Koppitz et al. (1997) hypothesize that stable, homogenous clones of P. australis are able to become established in given habitats because, in all probability, they are best adapted to those environments. This corroborates the work of Djerbrouni (1992) that certain P. australis isozyme variants seem to occur more commonly in particular environments. For the GC cluster, either the GC phenotype outcompeted virtually all other biotypes in the various habitats (differing in salinity and soil types) in the southern U.S. to the point of extinction, or the initial establishment of P. australis in this region involved a single genotype with broad ecological tolerances that has spread solely by vegetative propagation. We lean toward the latter hypothesis, but see the obvious need to test for other genetic and physiological differences among ramets from the various populations. This speculation of

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variation and distribution of the GC cluster does not preclude the possibility that the distribution of isozymic variation among other P. australis populations is adaptive (but see Section 4.5) In contrast to the Gulf Coast cluster, the two Mississippi River delta populations (MSD1 and MSD2) form their own highly differentiated cluster on the UPGMA although collected <10 km from where the Gulf Coast phenotype is common (Venice, LA; personal observation). The localization of the MSD1 and MSD2 phenotypes, and their distinctness from the Gulf Coast and other populations analyzed, provides strong support for the hypothesis that the MSD1 and MSD2 phenotypes which occur extensively throughout the MS River delta proper are probably introductions from outside the U.S. (Hauber et al., 1991). Aerial photographs of the delta appear to show that at least some of these populations were present as far back as 1940, suggesting that introduction occurred at least 60 years ago (Guerin and Hood, unpublished). Four of the five Midwest populations form a fairly distinct genetic cluster. Nevertheless, a high level of differentiation exists among the four populations, which have a mean genetic identity of 0.756. The distribution and differentiation among the Midwest populations is probably related to their phytogeographic history. It is possible they followed the retreating glaciers of the Pleistocene similar to the migration hypothesized for Ceratophyllum (Les, 1986). As suitable P. australis habitats became more isolated, gene flow was reduced among populations in those habitats leading to greater genetic differentiation. The distinct East Coast cluster including eight populations extending from South Carolina northeast of Charleston (SC1) to New Bedford, MA, (MA) are much less differentiated from each other (I ˆ 0.8714) as compared to the differentiation within the Midwest cluster. The greater genetic similarity is probably a consequence of the more stable and continuous coastal habitat allowing for a greater degree of migration and gene flow. This genetic evidence, and the report that P. australis remained unknown in South Carolina (and Georgia) until the mid-1970s (Statler, 1975) suggests that the reed has spread to the south along the Atlantic coast and that the establishment of at least some populations has been relatively recent. This is corroborated by our inability to find any populations in Georgia during collection trips along the Atlantic coast (personal observation). Although there is no direct evidence, it is known that marsh reed is commonly used in the construction of duck blinds. Hunters generally harvest their own culms for construction. These blinds, which are often left behind by the hunters, would provide ample opportunity for vegetative growth and subsequent colonization. This contributes another possible avenue for migration of Phragmites. A better understanding of the geographic patterns of distribution and differentiation of P. australis should be possible with a broader survey, including populations around the Great Lakes and southeastern Canada, particularly considering Phragmites occurs in a more or less continuous distribution from the upper Midwest to the Great Lakes and into Quebec. The degree of genetic similarity of the Quebec populations to the U.S. Eastern Seaboard populations would help in providing a complete picture of the pattern of colonization. Such studies should include collection of environmental data such as salinity, water depth, and soil type. A comprehensive survey using

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isozymes or random amplified polymorphic DNA (RAPD) markers of European populations and its comparison to a North American survey would be instrumental in gaining a clearer picture of geographic patterns of distribution and differentiation of P. australis. 4.5. The nature of invasive phenotypes of P. australis This study provides some initial insight into the nature of invasive phenotypes of P. australis in the United States. When comparing the described invasive phenotypes found in CT1 (B. Lapin, personal communication) and MSD1 (Hauber et al., 1991), there is clearly no genetic similarity pointing to a common origin. Considering these phenotypes individually, MSD1 is almost certainly an exotic type (or derivative) of unknown origin. CT1, on the other hand, shows relatively little distinctness (I ˆ 0.890) from the nonaggressive CT2 (B. Lapin, personal communication). CT1's isozymic similarity to other East Coast phenotypes casts doubt on the hypothesis that it represents a recent introduction unless one entertains the hypothesis that all current East Coast populations are recently introduced (Besitka, 1996). A population collected from Green Bay, WI, along the outskirts of Atkinson's marsh (WI3) occurs quite extensively along roads and in disturbed areas of the marsh. Though considered common to the area for over 45 years (Fassett, 1951), its coverage has certainly expanded dramatically since the construction of an interstate highway over the marsh in 1970, both in the marsh area and especially along road construction sites (G. Fewless, personal communication). Yet, the WI3 phenotype is not substantially differentiated from the other Midwest phenotypes which do not appear particularly invasive. A possible alternative explanation for the increased spread of P. australis in Atkinson's marsh and areas along the Eastern Seaboard is habitat manipulations and disturbances caused by humans (Marks et al., 1994). These include restrictions of tidal flow, increased sedimentation in marshes, increased road salt run-off, and increased nutrient run-off from farms and urban areas (Marks et al., 1994). These disturbances can create habitats where P. australis can compete more successfully than other macrophytes, thereby spreading rapidly in an invasive manner. To test whether human influence has played a primary role in the spread of P. australis, a more detailed history of human influence on particular wetlands would be needed with a record of the rate of spread of P. australis in these areas and the associated isozymic phenotypes. Performing common garden experiments with different isozymic phenotypes would also help to elucidate the influence of genetic differences. Acknowledgements We thank Steven P. Darwin, Beth Lapin, and David A. White for specimens and population information; Gary Fewless for background on Wisconsin populations; and especially David White, Wim van Vierssen, and two anonymous reviewers for comments on the manuscript. This research was funded, in part, by the Rev. John H. Mullahy Fund and a Loyola University Faculty Research Grant to Donald P. Hauber.

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