RESEARCH ARTICLE

Exploration of meaningful subspecies definitionsof the carnivorous Northern Pitcher Plant (Sarracenia purpurea),throughout its geographic distribution

Jennifer M. Karberg • Margaret R. Gale

Received: 15 January 2010 / Accepted: 11 August 2010 / Published online: 31 August 2010

� Springer Science+Business Media B.V. 2010

Abstract The carnivorous wetland plant, Sarracenia pur-

purea (the northern pitcher plant) is native to eastern and

Midwestern North America. This species is abundant within its

habitat but suitable habitat is increasingly scarce, raising

interest in S. purpurea restoration and conservation. Compli-

cating conservation planning, two controversial subspecies of

S. purpurea are historically defined primarily on morphologic

traits: S. purpurea subsp. purpurea distributed north of Mary-

land and S. purpurea subsp. venosa distributed south of

Maryland. S. purpurea is also found in three distinct habitat

types defined by substrate: acidic Sphagnum peatlands, acidic

sandy savannahs, and alkaline marl wetlands. In species level

studies, S. purpurea leaves have exhibited morphological

plasticity in response to environmental variability, bringing into

question the validity of subspecies definitions based on mor-

phology alone. This study examined morphologic and genetic

variation throughout S. purpurea’s natural distribution,

encompassing both the traditional subspecies and three unique

habitat types testing the validity of traditional subspecies defi-

nitions. Genetic analysis indicated possible ecological signifi-

cance of considering a new grouping of S. purpurea populations

into Midwest, N. East Coast and S. East Coast populations

(AMOVA % variation = 13.34, P = 0.0078) based on genetic

differentiation. Morphological variation in leaf shape mea-

surements supported this division as well as indicating plasticity

associated with environmental variables. This study conserva-

tively suggests that new, geographical area conservation units

may be a more important conservation unit for preserving

S. purpurea genetic variation and morphological plasticity than

traditional subspecies definitions.

Keywords Wetlands � Conservation genetics �Sarracenia purpurea � Subspecies definition � Carnivorous

plants

Introduction

Habitat alteration and fragmentation often function as

drivers of species declines and extinctions (Fahrig 2002;

Frankham et al. 2004). Conservation research explores

ways to successfully understand and plan ecosystem res-

toration, typically with the intent to restore or preserve a

particular endangered or threatened species. In plant con-

servation, the question arises as to which level to conserve a

species to gain the most beneficial conservation result: at

the entire species-level, subspecies-level or even at the

individual population level (Haig et al. 2006; Leadlay and

Jury 2006). In the United States, legal prioritization for

species restoration is controlled by the Endangered Species

Act, which allows the listing of ecologically important

subspecies and other groupings for federal protection (Haig

et al. 2006), yet no standardized definition of what consti-

tutes a plant subspecies exists. Traditionally plant subspe-

cies definitions have been primarily applied using one or

two morphological traits (Haig et al. 2006). A subspecies

designation can most often refer to geographic variation

present within a species (morphological response to

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10592-010-0123-7) contains supplementarymaterial, which is available to authorized users.

J. M. Karberg (&)

Science and Stewardship Department, Nantucket Conservation

Foundation, 118 Cliff Dr, Nantucket, MA 02554, USA

e-mail: jkarberg@nantucketconservation.org

M. R. Gale

School of Forest Resources and Environmental Science,

Michigan Technological University, 1400 Townsend Dr,

Houghton, MI 49931, USA

123

Conserv Genet (2010) 11:2369–2378

DOI 10.1007/s10592-010-0123-7

environmental characters within a particular geographic

region) or an evolutionary step (i.e. subspecies on the

evolutionary path to becoming individual species) within a

species (Hamilton and Reichard 1992). Each of these two

characterizations of plant subspecies often reflects impor-

tant ecological differences that many increase the prioriti-

zation for conservation needs. Understanding the ecological

relevance of a defined categorization for a particular plant

of concern can have great import on planning successful

conservation across a species’ range.

Carnivorous plants, plants that utilize digested nutrients

from insects for plant nutrition, are represented in many

biomes across the world with North America species

concentrated in nutrient poor wetland environments

(Christensen 1976; Givnish 1989; Schnell 2002). Sarrace-

niaceae, the pitcher plant family, is the most common

carnivorous plant family in North America (Schnell 2002).

Of the estimated nine species within the Sarracenia genus,

Sarracenia purpurea (the northern pitcher plant) represents

the most widespread pitcher plant, distributed from north-

ern Canada, through the Midwest and along the East Coast

to Florida (Schnell 2002). S. purpurea is a long-lived

perennial plant (35–50 years) reproducing both clonally

(spreading through rhizomes) and sexually (setting viable

seeds) (Schnell 2002). S. purpurea individuals are abun-

dant within their habitat yet that habitat is becoming

increasingly rare due to anthropogenic landscape devel-

opment and habitat alteration making conservation an

increasing concern, particularly in southern distributions

and in developing Midwestern areas (Schnell 2002).

A traditional and controversial definition of two sub-

species divides the distribution range of S. purpurea into

northern and southern subspecies with the geographic

dividing line around Maryland (Schnell 1979, 2002). These

two subspecies are defined exclusively based on mor-

phology with Sarracenia purpurea subsp. purpurea in the

north and Sarracenia purpurea subsp. venosa in the south

(Schnell 1979, 2002; Wherry 1933) (Fig. 1). The accuracy

of these subspecies definitions has been widely debated

(Bell 1949; Ellison et al. 2004). Four major morphological

characteristics distinguish between the subspecies: hairi-

ness of external leaf surface, flower color, hood lobe size,

and the ratio of leaf length to pitcher mouth width (Schnell

2002). The first two characteristics can be subjective while

the last two are quantitatively measured leaf shape traits. S.

purpurea leaves have exhibited morphological plasticity in

response to environmental characteristics; responding to

pH shifts, degrees of ecosystem wetness, precipitation, and

nutrient flux (Ellison et al. 2004; Karberg and Gale 2006;

Mandossian 1966) making leaf shape a questionable

character for distinguishing these subspecies. Recent work

examining morphological variation in populations through

the entire distribution range of S. purpurea showed no

support for the use of historic subspecies classifications in

explaining morphological variation (Ellison et al. 2004).

Observed morphological variation may be more related to

habitat/environmentally-induced plasticity than evolution-

ary differences between strictly defined subspecies.

A previous study has shown strong support for genetic

differentiation of S. purpurea subspecies (Godt and

Fig. 1 Distribution range of

Sarracenia purpurea indicating

geographical locations of

historic subspecies, S. purpureasubsp purpurea and S. purpureasubsp venosa, as well as

locations of isolated distinct

subspecies of S. purpurea not

included in this study

2370 Conserv Genet (2010) 11:2369–2378

123

Hamrick 1999). This study attempted to examine genetic

differentiation between S. purpurea subsp purpurea and

three varieties of S. purpurea subsp venosa (vars. burkii,

montana, venosa) using only two sampled populations of

each. Observed genetic differentiation between S. purpurea

subsp purpurea and S. purpurea subsp venosa maybe be a

result of the inclusion of two very isolated and divergent

varieties (vars. burkii and montana), which are isolated

from the otherwise continuous distribution range of

S. purpurea. Examination of the genetic relatedness of just

S. purpurea subsp purpurea and S. purpurea subsp venosa

may show less strong differentiation. S. purpurea is pre-

dominantly a clonal species and tends to show moderate to

low levels of genetic diversity with fairly low genetic

differentiation between populations (GST ranging from

0.072–0.038) even across great distances (Karberg and

Gale 2006; Parisod et al. 2005; Schwaegerle and Schaal

1979) although Schwaegerle and Schaal (1979) found

increased levels of genetic differentiation between popu-

lations when sampling across the species range, not con-

sidering subspecies differentiation.

In addition to conflicting results from studies of mor-

phologic and genetic variation, Sarracenia purpurea

inhabits three environmentally distinct habitat types across

its range (Schnell 2002), perhaps contributing to observed

variation in leaf morphology and the observed genetic vari-

ation resulting from localized genetic adaptations. S. pur-

purea populations found within the historic northern

subspecies distribution can be located in two very different

habitat types. The typical habitat type in northern regions is

an acidic, fairly low nutrient, Sphagnum-dominated peatland

(Schnell 2002). The second habitat, found primarily around

the Midwestern Great Lakes region, is an extremely alkaline,

calcareous marl wetland (Schnell 2002). Only one habitat

type is typically found within the range of the southern

subspecies and it is an acidic peaty-sand savannah with lower

nutrient concentrations (Schnell 2002). No clear under-

standing exists about the effect of habitat type on morpho-

logic and genetic variation in S. purpurea. The interaction of

both habitat type as defined by substrate and geographic

distance between populations may be influencing observed

morphological and genetic variation that has previously been

attributed to a strict North/South subspecies definition.

The purpose of this study was to examine the genetic

structure of Sarracenia purpurea populations across the

species’ continuous range in an attempt to verify historic

subspecies definitions of S. purpurea subsp purpurea and S.

purpurea subsp venosa. Our goal was to utilize previously

understudied molecular markers (ISSR sequences) to define

populations in a way that site managers can easily replicate

in order to designate specific populations for conservation

planning, and select seed sources for restoration planning.

Thus our objectives in this paper were to (1) determine if

genetic and morphological patterns together substantiate the

traditional subspecies definition, and (2) if these definitions

do not hold, determine if a different subspecies definition for

S. purpurea can be defined to aid in designing successful,

economic, and efficient conservation.

Methods

Sample collection

Late summer 2006, populations of S. purpurea were sam-

pled across the species distribution range (Fig. 2) encom-

passing both the historic subspecies and the three habitat

types (marl, sand and Sphagnum). For each population

(n = 16), we sampled at least 15 randomly selected plants

(more if the population was larger), recording a suite of

morphological traits (leaf number, leaf length, leaf width,

keel width, leaf hairiness, pitcher mouth width, pitcher flap

width, flower number, flower height, flower color, basal

rosette diameter). From the measured plants, we randomly

collected leaf tissue samples from up to 15 individual

plants per site for genetic analysis (total n = 237); sample

number was less than 15 in some small populations (see

Table 4). Collected leaf tissues was cut from the hood of

the pitcher and rinsed with deionized water before being

transported to the lab. Field-collected tissue was stored at

-80�C until DNA extraction.

Leaf tissue DNA was extracted utilizing the Qiagen

DNeasy Plant Mini Kit and extracted samples were stored at

-20�C. Samples were screened with ISSR primers pur-

chased from the University of British Columbia Biotech-

nology Laboratory, allowing the selection of 13 reproducible

and polymorphic primers (Table 1). Subsequent primer

sequences, matching those screened, were purchased

through Invitrogen. Inter-Simple Sequence Repeats (ISSRs)

are highly reproducible, dominant molecular markers, cheap

to implement, and have been consistently employed in

assessing population genetic diversity and understanding

intraspecific relationships in plants (Roux et. al 2006; Wolfe

and Liston 1998; Zietkiewicz et al. 1994). ISSRs contain a

large number of polymorphisms allowing differentiation at

the intraspecies level and have proven useful in determining

phylogenetic relationships between populations, particu-

larly in species with some degree of clonality. The neutral

nature of ISSR markers makes reproducibility important for

increasing the strength of statistical analysis (Wolfe 1998).

During primer screening, each primer was independently

tested twice on five randomly selected representatives from

each population. Bands present in only one of the repeats

were eliminated from analysis. An examination of thirteen

primers allowed the analysis of 80 reproducible loci. After

primer selection (n = 13), each population (n = 16) was run

Conserv Genet (2010) 11:2369–2378 2371

123

through two independent Polymerase Chain Reactions

(PCRs) for each primer to reduce error that may occur during

PCR. Additionally, to further reduce the chance of proce-

dural errors, two negative controls were run with each PCR.

These precautions gave the authors strong confidence in the

accuracy of examined bands. Laboratory constructed PCR

Master Mix was a 15 ll reaction mixture of 2 ll DNA in

solution, 1 ll of GoGreen Taq polymerase in solution, 2.5 ll

of reaction buffer, 1.9 ll of MgCl2, 0.25 ll of dNTPs, 6.35 ll

of double-distilled water, and 1 ll of primer. The PCR

thermocycler program consisted of an initial denaturing step

of 4 min at 94�C followed by 1 min at 72�C, 1 min at primer

optimal Tm8C (Table 1), and 1 min at 94�C for 45 cycles.

PCR product was then separated on a 1% agarose electro-

phoresis gel, with a 1 kb ladder, stained in ethidium bromide

and visualized using a digital camera.

Sample analysis

A principal component analysis (PCA) based on a Pearson’s

correlation matrix was constructed using sampled mor-

phological measurements and transformations (flower

number, flower height, plant basal diameter, leaf number,

leaf length, leaf hairiness, pitcher mouth width ratio of

pitcher length to pitcher mouth width and ratio of keel width

to leaf width) to determine the characteristics that explained

the majority of variation observed between individual

populations. Two measured morphological characters

Fig. 2 Population sample

locations across the range of

Sarracenia purpurea. State-

level grouping circles indicate

assigned geographic area

classification (Midwest, North

East Coast, and South East

Coast). Population marker

shapes indicate substrate type

(filled circle Marl, filled starSand, and filled triangleSphagnum sites)

Table 1 Inter simple sequence repeat (ISSRs) primer sequences and

Tm �C selected for population analysis

Primer name Sequence: (50–30) Concentration Tm�C

830 TGTGTGTGTGTGTGTGG 274 52

836 AGAGAGAGAGAGAGAGYA 179 53

842 GAGAGAGAGAGAGAGAYG 186 55

850 GTGTGTGTGTGTGTGTYC 260 55

851 GTGTGTGTGTGTGTGTYG 262 55

854 TCTCTCTCTCTCTCTCRG 298 55

864 ATGATGATGATGATGATG 265 48

874 CCCTCCCTCCCTCCCT 255 56

880 GGAGAGGAGAGGAGA 167 48

872 GATAGATAGATAGATA 224 40

826 ACACACACACACACACC 250 52

2372 Conserv Genet (2010) 11:2369–2378

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consistently explained the majority of observed variation

among populations, flower height and leaf length.

Examination of population groupings from the principal

component analysis suggested the establishment of a geo-

graphical area grouping of the data for statistical analysis in

addition to the a priori establishment of groupings by his-

toric subspecies and substrate type. Three geographical

areas were defined as (1) Midwest, (2) Northern East Coast,

and (3) Southern East Coast (Fig. 2).

A one-way analysis of variance (ANOVA) examined the

differences of the two characters (flower height and leaf

length) as partitioned within four population groupings: (1)

all populations; (2) northern and southern subspecies; (3)

three substrate types—marl, sand, Sphagnum; and (4)

geographical area—Midwest, N. East Coast, and S. East

Coast populations. We also examined a one-way analysis

of variance (ANOVA) of the ratio of leaf length to pitcher

mouth width and the ratio of keel width to leaf width, as

these transformations have shown a strong relationship to

environmental variation, particularly pore-water nitrogen

concentrations (Ellison et al. 2004). A Tukey analysis of

mean differences examined partitioning of morphological

variability among the different population groupings.

Analysis of all morphological characters was conducted

using the R statistical package (Team RDC 2006).

For the molecular data, 16 populations were explored

using 13 reproducible ISSR primers. Polymorphic bands

were scored as 1 (present) or 0 (absent) for analysis. All

analyses assumed dominant, diallelic markers in Hardy-

Weinberg equilibrium (Lynch and Milligan 1994). Patterns

of genetic variation were examined through an AMOVA

(analysis of molecular variation). Four different AMOVAs

were explored: (1) all populations; (2) northern and

southern subspecies; (3) substrate type and (4) geographic

area. Genetic structure in the AMOVA was calculated from

Euclidian square genetic distance. Calculated genetic dis-

tances were also used to perform Mantel’s test of geo-

graphic distance (Meirmans and Van Tendering 2004).

AMOVAs were conducted using Arlequin ver. 3.0

(Schneider et al. 2005).

Using POPGENE ver. 1.31 (Yeh et al. 1997) we cal-

culated two genetic diversity measures on all populations,

Nei’s measure of genetic diversity (H = 1 – Rpi2) and

Shannon’s information index (I = Rpi log pi) where pi is

the frequency of alleles in a population (Lewontin 1972)

assuming Hardy–Weinberg equilibrium. We calculated

Nei’s GST (GST = (1 – (HS/HT)), a measurement of pop-

ulation differentiation, essentially the proportion of genetic

diversity among populations (Nei 1973). Presence and

absence of individual allele loci between populations were

examined for patterns of allelic differences between

populations.

Results

Morphological data

The principal component analysis identified two morpho-

logical characters that explained a significant portion of the

variation observed between populations; flower height

(explained 88.7% of variation in the first principal axis) and

pitcher leaf length (explained 9.7% of the variation in the

second principal axis (Table 2). These two variables as well

as the ratio of leaf length to pitcher mouth width and the ratio

of keel width to leaf width were analyzed in relation to four

different groupings. The means for the four morphological

characters were significantly different by geographic area

and substrate type but not by subspecies definition (Fig. 3).

Within the geographic area classification, the Midwest

and S. East Coast populations had significantly different

flower heights, leaf/mouth ratios and keel width ratios, but

Midwest and N. East Coast had significantly different leaf

lengths (Fig. 3). Examining morphological traits grouped

by substrate type (Sphagnum, marl, sand) proved signifi-

cant (Fig. 3). Sphagnum and sand sites had significantly

taller flowers than marl sites. Marl and sand sites had

significantly shorter leaf lengths compared to Sphagnum

sites and Sphagnum and marl sites had significantly dif-

ferent leaf/mouth ratios and keel width ratios from sand

sites. Substrate type and geographical area significantly

influenced both flower and leaf shape morphology.

Genetic data

AMOVAs performed on four groupings (individual popu-

lations, subspecies, geographic area, and substrate type)

yielded results both similar and different from the mor-

phological analysis (Table 3). Within all AMOVA analy-

ses, the majority of genetic variation was significantly

Table 2 Principal component analysis of morphological and envi-

ronmental variables sampled in all populations (n = 16)

Variable Eigenvalues

Principal component axis 1

Proportion of variance 88.70%

Cumulative proportion 88.70% Flower height 0.9803

Standard deviation 69.033

Principal component axis 2

Proportion of variance 9.70%

Cumulative proportion 98.40% Leaf length -0.9528

Standard deviation 22.823

Two axes explained 98.4% of the variation using two variables:

flower height and leaf length

Conserv Genet (2010) 11:2369–2378 2373

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located within individual populations, with only small but

significant amounts of observed variation explained by

population groupings (% variation = 47.08, P \ 0.0001).

The AMOVA grouping of populations by substrate type

was not significant (P = 0.273) for among group variation,

showing little genetic variation among the populations

located on the different substrate types. The AMOVAs for

subspecies type (P = 0.029) and geographic area

(P = 0.008) were both significant, with the geographic

area grouping explaining only slightly more variation

among populations (% variation = 13.34). A Mantel test

using Nei’s genetic distance showed no significant relation

Geographic

Ave

rage

Flo

wer

Hei

ght (

cm)

0

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Ave

rage

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gth

(cm

)

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Rat

io L

eaf L

egnt

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itche

r

Mou

th M

idth

0.00

0.05

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0.30

Rat

io K

eel W

idth

/Lea

f Wid

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0.0

0.1

0.2

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0.4

0.5

Substrate

Ave

rage

Flo

wer

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ght (

cm)

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20

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rage

Lea

f Len

gth

(cm

)0

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Midwest N. East Coast S. East Coast

Midwest N. East Coast S. East Coast

Midwest N. East Coast S. East Coast

Midwest N. East Coast S. East Coast

Sphagnum Marl Sand

Sphagnum Marl Sand

Sphagnum Marl Sand

Sphagnum Marl Sand

Rat

io K

eel W

idth

/Lea

f Wid

th

0.0

0.1

0.2

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ba/b

a

aa

aa

aa

aa

a/ba/b

a/b

a/b

bb

b bb

b b b

b

A E

B F

C G

HD

Fig. 3 Means of four morphological characters (flower height, leaf

length, ratio leaf length/pitcher mouth width, and ratio keel width/leaf

width) organized by geographic area classifications (Midwest, N. East

Coast, S. East Coast) (A–D) and by substrate type (Sphagnum, Marl,

Sand) (E–H). Letters indicate significant differences between means,

bars represent standard errors

2374 Conserv Genet (2010) 11:2369–2378

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between increasing geographic distance and increasing

genetic distance indicating that geographic distance

between populations does not explain observed patterns in

genetic variation (r = 0.182, P = 0.108).

Genetic diversity measures of all populations showed

moderate levels of genetic diversity within most populations

(Table 4), consistent with results seen in previous studies of

S. purpurea (Godt and Hamrick 1999; Karberg and Gale

2006; Parisod et al. 2005). Nei’s measure of genetic diver-

sity (H = 1 – Rpi2) ranged from 0.236 - 0.3301, with

Midwest Sphagnum populations having the most diversity.

Shannon’s information index (I = Rpi log pi) ranged from

0.364 - 0.501, again with the Midwest Sphagnum popula-

tions having the most diversity followed by the N. East

Coast populations and then the S. East Coast populations.

GST ranged from 0.320 in Midwest sites to 0.238 in N. East

Coast sites and 0.186 in S. East Coast sites. This indicates

that genetic differentiation among Midwest populations is

higher compared to S. East Coast and N. East Coast popu-

lations, although GST numbers are low across all popula-

tions. Looking at allele loci variation among the three

Geographic areas, the Midwest populations contain all but

one allele loci, which is only found within the S. East Coast

populations. The N. East Coast and S. East Coast popula-

tions both lack the same six alleles which are present in the

Midwest populations, marking an increased genetic relation

between these populations compared to the Midwest

populations.

Table 3 Analysis of molecular

variation (AMOVA) for four

different population groupings:

(1) All sampled populations,

(2) Subspecies—Sarraceniapurpurea subsp. purpurea and

Sarracenia purpurea subsp.

venosa, (3) Substrate

Types—Sphagnum, Marl,

Sand and (4) Geographical

area—Midwest, North East

Coast, South East Coast

Grouping Source of variation df Percent

variation

P

All populations Among populations 15 47.08 \0.001

Within populations 221 52.92 \0.001

Subspecies Among groups 1 12.6 0.02933

Among populations within groups 14 38.99 \0.001

Within populations 221 48.41 \0.001

Substrate Among groups 2 2.11 0.2727

Among populations within groups 13 45.38 \0.001

Within populations 221 52.21 \0.001

Geographical Among groups 2 13.34 0.0078

Among populations within groups 13 37.21 \0.001

Within populations 221 49.45 \0.001

Table 4 Genetic diversity and

population genetic indices for

each populations and by

Geographic area (in bold)

H = Nei’s genetic diversity

index = (1 – Rpi2) where pi is

the frequency of the ith allele,

I = Shannon’s diversity

index = Rpi log pi),

GST = Nei’s coefficient of gene

variation = (1 - Hs/HT) where

Hs is within populations and HT

is the entire population

Site code Site location n H I % Polymorphic

loci

Total H Total I GST

Midwest

AT Alger Co., MI 15 0.25 0.34 76.25

HN Alger Co., MI 15 0.25 0.37 70

JA Bayfield Co., WI 15 0.27 0.41 75

SP Bayfield Co., WI 15 0.26 0.39 76.25

PN Porter Co., IN 15 0.25 0.38 73.75 0.33 0.5 0.32

LO Keweenaw Co., MI 15 0.25 0.37 66.25

JB Stark Co., MI 15 0.18 0.28 52.5

MI Mackinaw Co., MI 15 0.21 0.31 60

RR Presque Isle Co., MI 15 0.2 0.3 57.5

US Mackinaw Co., MI 13 0.18 0.28 50

TS Ontario CA 15 0.16 0.25 53.75

N. East Coast

BG Kennebec Co., ME 15 0.17 0.26 53.75 0.25 0.38 0.24

TM Worcester Co., MA 15 0.22 0.32 62.5

S. East Coast

NJ Ocean Co., NJ 15 0.19 0.3 62.5

GS Brunswick Co., NC 14 0.22 0.33 66.25 0.24 0.36 0.19

LC Marlboro Co., SC 15 0.17 0.26 56.25

Conserv Genet (2010) 11:2369–2378 2375

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Discussion

This study attempted to examine the validity of an historic

subspecies description of Sarracenia purpurea utilizing

surveys of both morphologic and genetic variation within

and among sampled populations. Plant morphology has

historically been used to define subspecies of S. purpurea but

morphological characters observed in this study showed no

significant support for a subspecies classification dividing

S. purpurea into a northern subspecies (S. purpurea subsp.

purpurea) and a southern subspecies (S. purpurea subsp.

venosa) as described by Schnell (2002). Morphological

characters in this species respond to environmental variation

and potentially localized adaptation and cannot be used

alone to develop subspecies designations for S. purpurea.

Morphological variation in the ratio of leaf length to pitcher

mouth width (a term used in the differentiation of historic

subspecies) showed significant differences in population

grouping by both geographic area and substrate type (Fig. 3)

but not historic subspecies. Examined morphologic traits

support the classification of populations based on geographic

area, significantly distinguishing Midwest populations and

S. East Coast populations. These regions have very different

environmental trends from annual temperature to annual

precipitation. Morphologic variation observed within sub-

strate types classifications indicated significant differences

separating Sphagnum and marl wetlands from the southern

sand wetlands (Fig. 3). These substrate types can have very

different environmental characteristics from soil pH to soil

moisture. The relation of morphological variation to these

two population groupings supports the idea that S. purpurea

morphological variation is closely related to environmental

variation as described by Ellison et al (2004) and not a

realistic or reliable measure of subspecies distinction.

Population genetics, on the other hand showed support

for both a geographical grouping and the historic subspe-

cies grouping. Populations sampled across the entire range

of this species appear closely related genetically with only

a few loci separating populations. The moderate amounts

of observed genetic variation within populations and

AMOVA analyses indicate strong genetic similarity

between all sampled populations (Table 3), which is

expected given the clonally reproductive nature of this

plant. Even though populations are genetically very similar

to each other, we were able to detect small but significant

amounts of variation explained by grouping populations

into the defined geographic areas and by historic subspe-

cies with no genetic support for a substrate type grouping

(Table 3). Examination of morphologic traits in addition to

genetic variation is important when using a neutral marker

system such as ISSRs, which do not illustrate direct trait

adaptation but rather overall genetic patterns (Wolfe 1998;

Wolfe and Liston 1998). Genetic support may exist for the

subspecies classification but strict morphological descrip-

tion cannot be used to assign populations to a particular

subspecies grouping due to the plastic response of plant

morphology to environmental variation. This makes the use

of a geographic grouping, supported by both morphology

and genetic variation, potentially better for classifying all

variation that exists in S. purpurea and helping prioritize

conservation efforts. The geographic grouping accounts for

both environmental variation and historic distributions of

S. purpurea that may influence genetic patterns observed in

modern population distributions.

The new proposed organization of populations into

geographic areas, supported by both observed genetic

diversity and morphological variation, could result from a

few historical scenarios. An historic separation between

these three geographic areas (Midwest, N. East Coast and

S. East Coast) could, through an extended period of low

gene flow, have lead to small amounts of genetic differ-

entiation and local adaption to the unique habitat charac-

teristics present in each geographic area. Historically the

entire range of S. purpurea north of Maryland was glaci-

ated as recently as 10,000 years ago while southern pop-

ulations were never glaciated (Dorr and Eschman 1970).

Two scenarios are often proposed for historical reestab-

lishment of S. purpurea populations in glaciated areas,

(1) source populations were remnant, isolated populations

within the glaciated S. purpurea range, or (2) source pop-

ulations came from the southern, unglaciated populations

(Shehlik et al 2002). Either of these reestablishment

methods could explain observed variation among these

geographical groups. Additionally, the three substrate types

examined in this study exhibit a range of different localized

variation in pH, hydrology, water chemistry etc. Genetic

and morphological variation among geographic areas may

be influenced partially by localized responses to substrate

type. No statistical genetic support exists for classifying

populations based on substrate type alone but morpholog-

ical differences observed in different substrate types may

be recent with genetic differentiation not yet occurring

within these populations. On the other hand S. purpurea

may inherently have enough genetic variability to allow

morphological plasticity without necessitating genetic dif-

ferentiation, indicating that observed genetic variation

within the geographic grouping is related to historic

S. purpurea ranges.

For Sarracenia purpurea, utilizing a geographic area

subspecies definition may be a more ecologically important

approach to classify within-species variation, than the his-

toric subspecies definition. S. East Coast populations contain

significantly different genetic and morphological variation

in comparison to N. East Coast and Midwest populations,

which are genetically significantly differentiated from each

other. A useful classification to encompass unique variation

2376 Conserv Genet (2010) 11:2369–2378

123

within populations would consider the S. East Coast popu-

lations one conservation unit, the Midwest sites another unit

and the N. East Coast wetlands the last conservation unit,

with special consideration given to localized morphologies

expressed in response to different substrate types. Both the

S. East Coast sand savannas and Midwest marl substrate

systems are declining across their range through habitat

destruction indicating an increased need to plan successful

restoration in these geographic regions.

Analysis of genetic variation indicated that even though

geographic area groupings significantly explained variation

among populations, a significant portion of observed

genetic variation was located within individual populations

(% variation = 49.45, P \ 0.0001). Effective restoration

of plant variation (genetic and morphological) would

therefore benefit from increased preservation of individual

populations.

Conclusions

Our results did not support the traditional definition of two

distinct subspecies of Sarracenia purpurea following a

strict north/south division. Instead, our study supports a

more ecologically important designation of a geographic

area based subspecies definition delineating populations of

S. purpurea into three categories: S. East Coast, N. East

Coast and Midwest (Fig. 2). These categories represent

areas containing the majority of genetic and morphologic

variation represented by the species. Our analyses showed

significant differences in genetic variation and morphology

between the three geographic areas but the ecological

reasons for this variation are not well understood. S. pur-

purea leaves may show increased morphological plasticity

in response to environmental variation. In order to consider

how much of our field observed variation is due to envi-

ronmental conditions and not genetics, intensive common

garden and greenhouse experiments will need to be con-

ducted. Additionally, due to the scarcity of populations in

the southern portion of the S. purpurea range, sample sizes

were small for both the S. East Coast geographic grouping

and the S. purpurea subsp venosa grouping. Increased

sampling in these populations may clarify differences in

morphological versus genetic variation observed in this

study.

Populations of a species can be differentiated based on

geographic segregation, acknowledging the theory that

differences between geographic areas, environmental

characteristics and increased distance leading to lower gene

flow, may significantly alter the genetic and morphological

make up of a species. Using this new subspecies definition,

geographical units are important when they contain some

characteristics, morphological and/or genetic, that are

unique to their population, increasing their conservation

concern. This definition might be more appropriate for

individual plant species relating to evolutionary theories of

plant population dynamics. The federal Endangered Spe-

cies Act allows the recognition of plants of conservation

concern at a subspecies level, making accurate designations

of subspecies, as in the case of Sarracenia purpurea vital

for planning economical and successful restoration.

Acknowledgments The authors which to thank the staff and

supervisors at Isle Royale National Park, South Carolina State Parks,

The Nature Conservancy of North Carolina, Harvard Forest, Jackson

Bog Ohio, Indiana Dunes National Lakeshore, Pictured Rocks

National Lakeshore, Apostle Islands National Lakeshore, and Leba-

non State Forest New Jersey for logistical support. Dr. Joy Marburger,

Dr. Christopher Webster, Dr. Leah Vucetich, and Dr. Sarah Green for

assistance in project development. This research was supported in part

by grants from the National Park Service, McIntire-Stennis, the

DeVlieg Foundation, and Michigan Technological University.

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