Chromosomes tell half of the story: the correlation
between karyotype rearrangements and genetic diversity
in sedges, a group with holocentric chromosomes
ANDREW L. HIPP,*† PAUL E. ROTHROCK,‡ RICHARD WHITKUS§ and JAIME A. WEBER*
*The Morton Arboretum, 4100 Illinois Route 53, Lisle, IL 60532-1293, USA, †The Field Museum, 1400 S. Lakeshore Drive,
Chicago, IL 60605, USA, ‡Taylor University, Randall Environmental Studies Center, Upland, IN 46989-1001, USA,
§Department of Biology, Sonoma State University, Rohnert Park, CA 94928-3613, USA
Chromosome rearrangements may affect the rate and patterns of gene flow within
species, through reduced fitness of structural heterozygotes or by reducing recombi-
nation rates in rearranged areas of the genome. While the effects of chromosome
rearrangements on gene flow have been studied in a wide range of organisms with
monocentric chromosomes, the effects of rearrangements in holocentric chromo-
somes—chromosomes in which centromeric activity is distributed along the length of
the chromosome—have not. We collected chromosome number and molecular genetic
data in Carex scoparia, an eastern North American plant species with holocentric
chromosomes and highly variable karyotype (2n = 56–70). There are no deep genetic
breaks within C. scoparia that would suggest cryptic species differentiation. However,
genetic distance between individuals is positively correlated with chromosome number
difference and geographic distance. A positive correlation is also found between
chromosome number and genetic distance in the western North American C. pachy-
stachya (2n = 74–81). These findings suggest that geographic distance and the number of
karyotype rearrangements separating populations affect the rate of gene flow between
those populations. This is the first study to quantify the effects of holocentric
chromosome rearrangements on the partitioning of intraspecific genetic variance.
Keywords: amplified fragment length polymorphisms, Carex, chromosome rearrangements,
Cyperaceae, holocentric chromosomes, karyotype evolution
Received 15 January 2010; revision received 24 March 2010; accepted 26 March 2010
Chromosome rearrangements play an important role in
partitioning genetic variance, both within and among
species (White 1969; Rieseberg 2001; 2007; Noor et al.
2002; Ayala & Coluzzi 2005; Feuk et al. 2005; Noor et al.
2007). Although many studies have focused on the
role of inversions in protecting species-specific or
population-specific regions of the genome from recombi-
nation and thus preserving adaptive gene combinations,
chromosome fission and fusion also have the potential to
limit gene flow and drive speciation (Baker & Bickham
1986; Basset et al. 2006). Most work on the effects of
chromosome fission and fusion has been undertaken in
organisms that exhibit Robertsonian rearrangements
(Bantock & Cockayne 1975; Searle 1986; Davisson &
Akeson 1993; Nachman & Searle 1995; Hauffe & Searle
1998; Bidau et al. 2001; Pardo-Manuel de Villena &
Sapienza 2001; Rowell et al. 2002; Dumas & Britton-
Davidian 2002; Panithanarak et al. 2004), i.e. non-
reciprocal translocations involving fission and fusion at
or near a centromere. These studies demonstrate the
potential for Robertsonian fusions to decrease recom-
bination in rearranged areas of the genome among
populations that are connected by gene flow.
The effects of fission and fusion on gene flow
inorganismswhosechromosomes lack localized
Correspondence: Andrew L. Hipp, Fax: +1 630 719 2433;
? 2010 Blackwell Publishing Ltd
Molecular Ecology (2010)doi: 10.1111/j.1365-294X.2010.04741.x
centromeres—holocentric chromosomes—is not as well
understood. In holocentric chromosomes, spindle fibers
(microtubules) attach along the entire length of the
chromosome arm, dragging the chromosome broadside
toward the poles at anaphase (Dernburg 2001; Nagaki
et al. 2005). Chromosome fragments that would be
acentric and consequently lost in an organism with
localized centromeres may be inherited in Mendelian
fashion in organisms with holocentric chromosomes
(Faulkner 1972; Lucen ˜o 1993), and gametes involving
chromosome fragments are consequently expected to be
viable. Holocentric chromosomes are known in plants
primarily from the angiosperm sedge family Cyperaceae
(c. 5000 species) and its sister family, the rushes (Junca-
ceae, c. 430 species), but they are also known in at least
four other angiosperm genera, a few algae, several
arthropod orders, and nematodes, including the model
system Caenorhabditis elegans (Godward 1954; King 1960;
Flach 1966; Tanaka & Tanaka 1977; Sheikh et al. 1995;
Pazy 1997; Perez et al. 1997; Buchwitz et al. 1999; Nok-
kala et al. 2002; Guerra & Garcı ´a 2004; Wang & Porter
2004). Although strong selection against structural het-
erozygotes appears to maintain karyotypic stability in
C. elegans (Dernburg 2001), holocentry is accompanied
by extensive and rapid karyotypic variability within
and among species in the sedge genus Carex and in
some arthropod genera (e.g. Heilborn 1924; Hoshino
1981; Normark 1999; Cook 2001; Kandul et al. 2007). In
Carex, extensive studies of chromosome pairing relation-
ships in meiosis show an abundance of univalent and
hetermorphic trivalent associations, with quadrivalents
less common. The abundance of univalents and triva-
lents suggests that chromosome number changes are
due to breakages, fusions, or translocations (Wahl 1940;
Faulkner 1972; Hoshino 1981, 1992; Hoshino & Okam-
ura 1994; Hoshino & Onimatsu 1994; Hoshino et al.
1994). Moreover, these findings suggest that pairing
relationships are often not adversely affected by these
rearrangements. It is consequently unclear what effects
we should expect holocentric chromosome rearrange-
ments to have on gene flow within species.
In this study, we use a combination of molecular
genetic data(amplified fragment
phisms, AFLPs) and chromosome counts in the wide-
spread and karyotypically diverse sedge Carex scoparia
Schkuhr ex Willdenow var. scoparia (2n = 56–70) to test
two interrelated hypotheses: (i) that the various chro-
represent chromosome ‘races’ that are genetically differ-
entiated from one another to a degree comparable to
species or infraspecific taxa and (ii) that chromosome
rearrangements have an effect on molecular genetic
structure within species. If chromosome ‘races’ repre-
sent cryptic species, we expect to see deep genetic
breaks that correspond to chromosome rearrangements.
Similarly, if chromosome rearrangements restrict gene
flow, we expect to see a correlation between chromo-
some number differences and genetic distance at least
at small geographic scales. We compare our results with
a reanalysis of isozyme and chromosome number data
gathered in the western North American species Carex
pachystachya (Whitkus 1988b, 1991, 1992), which is
found in a different Carex clade, to investigate whether
our findings are unique to C. scoparia or applicable to a
broader phylogenetic range of the genus.
Materials and methods
The eastern North American C. scoparia var. scoparia
exhibits substantial karytotypic diversity, ranging from
2n = 56 to 2n = 70 (Fig. 1; Table 1; note that the
2n = 56 count has not been confirmed through our own
work, and no populations of this count are conse-
quently represented in the current study). A sister vari-
ety, C. scoparia var. tessellata Fernald & Wiegand, is a
regional endemic apparently limited to two counties in
Maine with only one known chromosome number
(2n = 68; Table 1). The western North American C.
pachystachya Chamisso ex Steudel is widely distributed,
ranging from northern California and Colorado to
Alaska. These species are in different major clades of a
species-rich and karyotypically diverse group, Carex
section Ovales, which has been the focus of substantial
phylogenetic and cytological research (Whitkus 1988a,b,
1991; Rothrock & Reznicek 1996, 1998, 2001; Reznicek &
Rothrock 1997; Hipp 2007; Hipp et al. 2006, 2007).
Detailed investigations in the genus have demonstrated
that hybrids tend to be either rare and sterile (Cayou-
ette & Catling 1992; Waterway 1994; Ball & Reznicek
2002; Smith & Waterway 2008), or frequent in a limited
number of taxa (Cayouette & Catling 1992). In section
Ovales, there is no demonstration of well established,
naturally occurring hybrids. All available information
on reproductive isolation between species indicates that
chromosomal differences do not play a role, but instead,
genetically determined barriers, either pre- or post-
mating, are the norm (Whitkus 1988a,b, 1991, 1992).
Previous study of C. pachystachya demonstrated that
populations are typically invariant in chromosome
number, and that when there is variation (in 3 of 20
populations studied), individuals differ by at most
one chromosome pair (Whitkus 1991). Variation within
sibling families has also been observed; variants all
possessed the same inferred diploid count, differing
only in the possession of tetravalents at meiosis I, which
is evidence of rearrangements (e.g. translocations or
2 A. L. HIPP ET AL.
? 2010 Blackwell Publishing Ltd
inversions) that affect pairing relationships but not
chromosome number (Whitkus 1991). Polyploidy is
unknown in the section (Whitkus 1988a, 1991; Rothrock
& Reznicek 1998), with the possible exception of a puta-
tive allotetraploid not closely related to C. scoparia
(Hipp et al. 2006). All observations of chromosome pair-
ing relationships in the genus support the stance that
chromosome evolution in Carex is predominantly by fis-
sion and fusion (reviewed in Hipp et al. 2009).
Sampling and chromosome data
Examplars were collected from 35 populations of C.
scoparia var. scoparia (N = 42 individuals) and two popu-
lations of C. scoparia var. tessellata (N = 6), a total of 48
individuals(Table 1;Fig. 1).
designed to maximize population sampling, based on
previous studies finding that genetic and karyotypic
variance are extremely limited within populations of
related Carex species in the section Ovales (Whitkus
1991, 1992). Additional genetic sampling within popula-
tions would very likely reduce sampling error in esti-
mating genetic distances among populations, but was
not conducted because the initial study of chromosome
variance from which our samples were drawn was not
built around a population-genetic study. Two species
(C. longii and C. vexans) were included as outgroups
based on previous phylogenetic studies (Hipp et al.
2006, 2007). Because analyses were conducted at the
level of the individual, on the assumption that individ-
uals are an appropriate proxy for populations, C. scopa-
ria var. scoparia specimens were subsampled to a single
individual per population for most analyses (referred to
as unique-by-population samples; N = 35 individuals,
24 permutations based on removal of one individual
per permutation from each of populations 3485, 3633,
and 3656 and removal of two individuals from 3489).
Analyses were conducted over all unique-by-population
mean ± standard error of the mean (SEM) calculated
over permutations. Carex pachystachya samples were
analysed at the population level (average population
size N = 17.4 ± 3.5 [SEM]), and consequently no sub-
sampling was necessary. Latitude and longitude were
estimated for each population to < 1 km precision by
mapping populations in Google Earth (Google, Inc.,
Mountain View, CA, USA). These coordinates were
used to generate a pairwise geographic distance matrix
of great-circle distances using the Haversine formula
Chromosome analyses for C. scoparia followed the
described by Rothrock & Reznicek (1996). For each indi-
vidual studied, an average of five pollen mother cells
were inspected at first meiotic metaphase. Drawings,
photographs (Fig. 2), and voucher specimens have been
& Morrison(1967), as
–75˚ –80˚–85˚ –90˚–95˚
64 × 2
64 × 2
68 × 4
66 × 2, 67
68 × 2
Fig. 1 Map of sites sampled, with inferred diploid chromosome counts. Multiplication sign (‘·’) indicates that multiple individuals
of the same count were found at a site. The two sites where C. scoparia var. tessellata was collected are indicated with open circles;
all other sites were collections of C. scoparia var. scoparia exclusively.
CHROMOSOMES AND GENETIC DIVERSITY IN SEDGES 3
? 2010 Blackwell Publishing Ltd
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This study began as a survey of chromosome diversity in
C. scoparia by Paul Rothrock and of genetic and chromosome
diversity in C. pachystachya by Richard Whitkus. Andrew
Hipp’s research addresses the diversification of flowering plant
lineages and traits, primarily in sedges and oaks. Paul Roth-
rock’s research focuses on the taxonomy of Carex section
Ovales, a diverse New World clade with numerous regional
endemics. Richard Whitkus researches the systematics and
evolutionary genetics of plants and applications of molecular
markers to mapping, diversity, and evolution. Jaime Weber
works on quantitative and molecular genetics of cultivated and
CHROMOSOMES AND GENETIC DIVERSITY IN SEDGES 15
? 2010 Blackwell Publishing Ltd