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Cryptic speciation in allotetraploids: Lessons from the Botrychium matricariifolium complex

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Premise of the study: Cryptic species are a challenge for botanists and taxonomists. To improve species delineation in the genus Botrychium (Ophioglossaceae), which includes multiple instances of allotetraploid speciation, we examined a cryptic species complex using genetics and morphology. Methods: We sampled species in the B. matricariifolium complex, concentrating on the Upper Peninsula of Michigan and including multiple proposed morphospecies. We analyzed over 1500 samples using 10 enzyme systems, measured 42 quantitative and qualitative morphological characters for over 650 individuals, and analyzed 145 samples using AFLPs. We tested for diagnostic enzymes in the morphospecies and calculated the correlation between morphological and genetic distances to determine whether putatively distinct morphotypes warrant taxonomic recognition. Key results: Allozyme allelic variation corresponded loosely to some morphotypes of B. matricariifolium, but with lower genetic distinction among them than found between B. matricariifolium and B. michiganense. Botrychium michiganense contains unique alleles, indicating a different hybrid origin from that of B. matricariifolium and supporting its status as a genetically distinct species. Conclusions: We showed that B. acuminatum morphology and genetics are accommodated taxonomically within B. matricariifolium; B. matricariifolium and B. michiganense likely represent hybridization events between related species; and morphotypes within B. matricariifolium likely represent repeated hybridization events between the same two parental species. These hybridizations have resulted in the array of morphotypes observed by field botanists. By helping to identify diagnostic morphological characters, genetic analyses also help us understand and resolve morphological variation observed in the field.
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AMERICAN JOURNAL OF BOTANY 103 (4): 1 – 14 , 2016; © 2016 Botanical Society of America 1
Cryptic species groups, which may contain undescribed species
within one historic taxonomic species, pose a problem for taxono-
mists, systematists, ecologists, and conservationists. Paris et al. (1989 ,
p. 47) de ned cryptic species using criteria provided by Stebbins (1950) ,
Grant (1981) , and Wiley (1981) as follows:
“1. Are poorly di erentiated morphologically.
2. Represent distinct evolutionary lineages because they are re-
productively isolated.
3. Have historically been misinterpreted as members of a single
Because cryptic speciation obscures morphological delineation
of taxa, researchers are likely to lump similar taxa that have distinct
lines of evolutionary history (in contrast to the splitting of a geneti-
cally cohesive species, which is likely when intraspeci c morpho-
logical variability equals or exceeds interspeci c variability). Paris
et al. (1989) used Botrychium subgenus Botrychium (= Botrychium
s.s.) as an example of cryptic species complexes. Within the genus,
B. matricariifolium provides an excellent case study in which cryp-
tic species and intraspeci c variability have long presented confusion
and controversy with regards to taxon delineation. Taxonomists
now have genetic tools to tease apart such cryptic species groups
and gain insight into their origins.
A clade comprising Botrychium , other members of Ophioglos-
saceae, and Psilotaceae is sister to Marattiales plus leptosporangiate
ferns ( Rai and Graham, 2010 ; Wickett et al., 2014 ; Rothfels et al.,
2015 ). Botrychium s.s. consists of about 35 distinct taxa (recognized
as species, subspecies, or varieties) worldwide, with a center of diver-
sity in northern North America ( Farrar, 2011 ). Closely related gen-
era include Botrypus Michx. (rattlesnake fern), Japanobotrychium
1 Manuscript received 12 June 2015; revision accepted 19 February 2016.
2 Chicago Botanic Garden, Department of Plant Biology and Conservation, 1000 Lake Cook
Road, Glencoe, Illinois 60022 USA;
3 Iowa State University, Department of Ecology, Evolution, and Organismal Biology, 253
Bessey Hall, Ames, Iowa 50011 USA; and
4 Tamarack Studios, P. O. Box 453, Manistique, Michigan 49854-0453 USA
5 Author for correspondence (e-mail:
Cryptic speciation in allotetraploids: Lessons from the
Botrychium matricariifolium complex 1
Evelyn W. Williams 2,5 , Donald R. Farrar 3 , and Don Henson 4
PREMISE OF THE STUDY: Cryptic species are a challenge for botanists and taxonomists. To improve species delineation in the genus Botrychium (Ophioglos-
saceae), which includes multiple instances of allotetraploid speciation, we examined a cryptic species complex using genetics and morphology.
METHODS: We sampled species in the B. matricariifolium complex, concentrating on the Upper Peninsula of Michigan and including multiple proposed
morphospecies. We analyzed over 1500 samples using 10 enzyme systems, measured 42 quantitative and qualitative morphological characters for over
650 individuals, and analyzed 145 samples using AFLPs. We tested for diagnostic enzymes in the morphospecies and calculated the correlation between
morphological and genetic distances to determine whether putatively distinct morphotypes warrant taxonomic recognition.
KEY RESULTS: Allozyme allelic variation corresponded loosely to some morphotypes of B. matricariifolium , but with lower genetic distinction among them
than found between B. matricariifolium and B. michiganense . Botrychium michiganense contains unique alleles, indicating a di erent hybrid origin from
that of B. matricariifolium and supporting its status as a genetically distinct species.
CONCLUSIONS: We showed that B. acuminatum morphology and genetics are accommodated taxonomically within B. matricariifolium ; B. matricariifolium and
B. michiganense likely represent hybridization events between related species; and morphotypes within B. matricariifolium likely represent repeated hybridiza-
tion events between the same two parental species. These hybridizations have resulted in the array of morphotypes observed by  eld botanists. By helping
to identify diagnostic morphological characters, genetic analyses also help us understand and resolve morphological variation observed in the  eld.
KEY WORDS allozymes; Botrychium matricariifolium ; cryptic species; ferns; Ophioglossaceae; polyploidy; speciation latest version is at
AJB Advance Article published on April 7, 2016, as 10.3732/ajb.1500281.
Copyright 2016 by the Botanical Society of America
Masam., and Sceptridium Lyon (grapeferns: Kato, 1987 ; Wagner
and Wagner, 1993 ; Hauk et al., 2003 ), all of which were previously
considered to be subgenera in the genus Botrychium s.l. ( Clausen,
1938 ). Of these genera, Botrychium s.s. has the smallest plants and
the most reduced morphology. Species are rarely taller than 20 cm,
with a leaf divided more or less equally into two segments, a spore-
producing sporophore and a photosynthetic nonfertile tropho-
phore ( Fig. 1 ). Stems are underground, upright, and usually
unbranched with a single apical bud (also underground) that pro-
duces a single aboveground leaf during the annual growing season.
Roots are  eshy and hairless. Nutrition is derived in part through
an underground association with mycorrhizal fungi of the genus
Glomus ( Winther and Friedman, 2007 ).
Sexual reproduction is via bisexual gametophytes (also under-
ground and supported by mycorrhizae) derived from spores re-
leased from aboveground leaves. Presumably due to the di culty of
sperm movement underground, new sporophyte production is al-
most exclusively by intragametophytic self-fertilization ( Soltis and
Soltis, 1990 ; Farrar, 1998 ; Barker and Hauk, 2003 ). Consequently,
because egg and sperm are derived by mitosis from the same hap-
loid spore, the resulting sporophytes are completely homozygous.
Occasional heterozygous individuals and hybrids attest to rare oc-
currences of cross fertilization between gametophytes, but predom-
inant intragametophytic sel ng and its consequences are a key part
of understanding the population biology of Botrychium .
Diploid species of Botrychium s.s. can be divided into two
groups based on morphology: section Lunariae that is once-pin-
nate with fan-shaped pinnae and section Lanceolatae that is
twice-pinnate with elongate pinnae ( Clausen, 1938 ).  is division
is supported by genetic analyses of plastid regions ( Hauk et al.,
2003 , 2012 ; Williams and Waller, 2012 ; Dauphin et al., 2014 ).
Molecular work also suggests the possible subdivision of section
Lunariae , with “Lunaria”, “Simplex”, “Pallidum”, and “Camp-
estre” subgroups variously recognized ( Hauk, 1995 , 2012 ; Wil-
liams and Waller, 2012 ).
More than half of Botrychium taxa are polyploid and unequivo-
cally derived through the process of allopolyploidy—hybridization
between genetically distinct diploid taxa accompanied by genome
doubling ( Hauk, 1995 , 2012 ; Farrar, 1998 , 2011 ; Hauk and Hau er,
1999 ; Williams and Waller, 2012 ).  e morphological distinction
among many of these polyploid taxa is subtle and presents serious
identi cation and management problems because some taxa are on
governmentally protected lists. Here we explore genetic and evolu-
tionary origins of problematic morphological variation in the B.
matricariifolium A.Braun complex of allopolyploid taxa in the
western Great Lakes (WGL) region, seeking explanations for ob-
served intra- and interspeci c variation and a consistent basis for
taxonomic recognition.  e complex exempli es the problems en-
countered with cryptic species groups; it is widespread and com-
mon in northeastern North America and exists in a frustrating
array of morphological forms.
Of the 17 formally named Botrychium species occurring in the
WGL region (nine diploids and eight polyploids), three formally
named species represent the B. matricariifolium complex: B. matri-
cariifolium , Botrychium michiganense W.H.Wagner ex A.V.Gilman,
Farrar and Zika, and B. acuminatum W.H.Wagner ( Wagner and
Wagner, 1990 , 1993 ; Farrar, 2011 ; Gilman et al., 2015 ). A fourth
member of the complex, B. hesperium (Maxon and Clausen) Wag-
ner and Lellinger is no longer recognized as occurring in eastern
North America ( Gilman et al., 2015 ), but has bearing on the origins
of the eastern taxa.
In an early genetic study of this complex in Michigan, Farrar
and Wendel (1997) introduced two putatively distinct taxa that
were originally identi ed by Henson and informally referred to
by Farrar and Wendel (1997) as B. matricariifolium “blocky”
morphotype and B. matricariifolium “acuminate” morphotype,
the later displaying intermediacy between B. acuminatum and
typical B. matricariifolium. In this study, we treat Wagner’s B.
acuminatum within the acuminate morphotype.  us, in the
WGL, four entities ( B. matricariifolium , B. matricariifolium
“blocky”, B. acuminatum , and
B. michiganense
) with similar and
to some degree overlapping morphologies are found that present
a bewildering array of intra- and interspeci c variability ( Fig. 2 ).
Collectively, plants of this complex are among the most common
ferns of the region.
Understanding similarities among species of the B. matricariifo-
lium complex requires consideration of their origins from diploid
Botrychium taxa.  e pinnate-pinnati d morphology of all taxa of
the B. matricariifolium complex is intermediate between that of
twice-pinnate members of section Lanceolatae and once-pinnate
members of section Lunariae , supporting a hybrid origin combin-
ing genomes of diploids from these two sections ( Fig. 3 ). Genetic
evidence from allozymes and plastid DNA strongly supports east-
ern B. angustisegmentum as the maternal plastid-donating parent
of eastern B. matricariifolium and B. acuminatum, whereas western
B. lanceolatum is indicated as the maternal parent of western
B. hesperium ( Wagner, 1993 ; Hauk and Haufler, 1999 ; Hauk
et al., 2003 ; Farrar, 2011 ; Williams and Waller, 2012 ). Allozyme
FIGURE 1 Morphology of a Botrychium fern, B. matricariifolium, with tro-
phophore, sporophore, pinnae, common stalk, and morphological mea-
surements made here, following Swartz and Brunsfeld (2002) ; see online
Appendix S3 for more information. Length measurements: (1) sporophore,
(2) sporophore branching point, (3) pinnae length, (4) pinnae width, (5)
rst pinnae pair, (6) trophophore, (7) second pinnae pair, (8) trophophore
width. Angle measurements: blue = pinnae stem angle, brown = pinnae
whole angle, maroon = pinnae partial angle, orange = 2nd pinnae pair
stem angle, green = 2nd pinnae pair partial angle. The circularity of basal
pinnae and the number of pinnae pairs were also measured.
evidence indicates that B. michiganense
in both eastern and western North
America combines alleles that are now
unique to either B. angustisegmentum
or B. lanceolatum ( Gilman et al., 2015 ),
suggesting an origin predating the dif-
ferentiation of these two Lanceolatae
Considering the most probable dip-
loid parents of the B. matricariifolium
complex within section Lunariae , and
drawing on evidence from codominant
allelic expression of metabolic enzymes
and morphology, Gilman et al. (2015)
concluded that the closest match for all
members of the complex is B. pallidum .
e morphological similarities of their
diploid ancestors ( B. pallidum × one of
the Lanceolatae species) likely contrib-
ute in large measure to morphological
similarities among extant members of
the B. matricariifolium complex. Species
bound aries are further confused by
the possibility that each allopoly-
ploid taxon was derived from multiple
hybridization events between the same
two diploid taxa, and from individuals
expressing intraspeci c morphological
variation.  e possibility of additional
cryptic species within B. matricariifo-
lium prompted our investigation. Here
we analyze complementary data de-
rived from allozymes, ampli ed frag-
ment length polymorphisms (AFLPs;
Vos et al., 1995 ), and morphometrics to
investigate morphotypes within the
B. matricariifolium complex in the
WGL region. Allozymes and AFLPs
allow intensive analyses of multiple
plants and populations based on nu-
clear genetic markers that re ect the
contributions from all parental taxa of
allopolyploid species. We expected that
observed genetic differences would
correlate with morphology within
groups and would help identify distinct
taxon units in the B. matricariifolium
complex. Our study includes analysis
of the possible genetic bases for the
“acuminate” and “blocky” morphotypes identi ed by Henson and de-
scribed by Farrar and Wendel (1997) .
Collections Collections examined in this study were made in 1995
and 1996 for a Botrychium diversity study sponsored by the Ottawa
National Forest and conducted by Henson and Farrar ( Farrar and
Wendel, 1997 ; Fig. 4 , open circles; Appendix S1, see Supplemental
Data with online version of this article). Additional relevant collections
FIGURE 2 Examples of the range of trophophore morphologies found within Botrychium matricariifolium,
including the “acuminate” and “blocky” morphotypes. These examples do not encompass all of the varia-
tion seen in B. matricariifolium , whose morphotypes can include immature individuals that resemble spe-
cies in section Lunariae . “EW” samples determined using AFLPs, and “DF” samples are determined by
documenting range-wide allozyme pro les of all Botrychium taxa
were made by Farrar and colleagues from 1997 through 2011. Williams
and Henson made collections in 2007–2009 ( Fig. 4 , closed circles;
online Appendix S2). Because of the di erent time periods of the
initial collections, the same individual plants could not be analyzed
for both allozyme and AFLP data ( Table 1 ). us, not all species
analyzed with AFLPs were analyzed with allozymes and vice versa.
Voucher specimens for allozyme-studied plants are maintained in
the Iowa State Herbarium. Voucher specimens for AFLP-studied
plants are deposited in the University of Michigan Herbarium. Scans
of all specimens are available from the authors.
Allozymes Samples for enzyme electrophoresis were cut from the
base of fresh leaf stalks, leaving the remainder of the leaf for press-
ing as a herbarium voucher. Electrophoresis procedures followed
Zika and Farrar (2009) . We analyzed 22 gene loci from 10 enzyme
systems using three bu er systems of Soltis et al. (1983) : bu er sys-
tem 7 (0.038 M LiOH, 0.188 M boric acid) for resolving enzyme
systems aspartate aminotransferase (AAT) and triose-phosphate
isomerase (TPI); bu er system 9 (0.065 M -histidine, 0.015–0.016
M citric acid, anhydrous) for resolving enzyme systems malate de-
hydrogenase (MDH), phosphoglucomutase (PGM), 6-phospho-
gluconate dehydrogenase (6-PGD), and phosphoglucoisomerase
(PGI); and bu er system 11 (0.4 M citric acid, trisodium salt) for
resolving enzyme systems aconitase (ACN), diaphorase (DIA),
isocitrate degydrogenase (IDH), and shikimate dehydrogenase
(SKDH). Numbered alleles for each locus were assigned relative to
their distance of migration from the origin, considering all species
of Botrychium ; lower numbers indicating faster migration. To fa-
cilitate discussion of genotypes of TPI-1 and PGI-1, we designated
FIGURE 3 Hypothesized relationships among the diploid and allotetraploid taxa discussed in this study.
Ploidy level is indicated by “2 n ” for diploid species (ellipses) and “4 n for derived allopolyploids (rectan-
gles). Trophophores of taxa in the left column exhibit twice-pinnate dissection whereas those of
Botrychium pallidum on the far right are once-pinnate. Trophophores of the allopolyploids, including
morphotypes of Botrychium matricariifolium , exhibit an intermediate condition referred to as pinnate-
pinnati d. Botrychium lanceolatum (“ancestral”) is a proposed extinct taxon that di erentiated into east-
ern B. angustisegmentum and western B. lanceolatum in North America.
FIGURE 4 Map of collection sites in northern Michigan (MI), Wisconsin,
and Minnesota (MN). Open circles are sites used for allozyme analysis
(collections from 1995 and 1996), solid circles are sites used for morpho-
logical and AFLP analyses (collected 2007–2009).
TABLE 1. Numbers of individuals per Botrychium taxon included in the study.
Sites of collection in the western Great Lakes region are shown in Fig. 4 .
Species AFLP
a Morphometrics
a Allozymes
B. angustisegmentum (2 n )36 67 43
B. lanceolatum (2 n ) 128
B. neolunaria (2 n ) 17 36 524
B. lunaria (2 n ) 5 3 465
B. matricariifolium (4 n ) 59 433 208
B. matricariifolium “blocky
3 3 100
B. matricariifolium “acuminate”
423 54
B. michiganense (4 n ) 5 42 115
B. minganense (4 n ) 5 23 287
B. pallidum (2n) 11 28 121
Total 145 658 2045
a Voucher information in online Appendix S2.
b Alloz yme numbers include representative plants from populations throughout the species
ranges, sampled in ongoing studies of the genus. Voucher information in online Appendix
S1. Speci c collection data are available in a database maintained by Farrar in the Iowa State
University herbarium ( Additional
species information, including species’ ranges, are available in the following publications:
Farrar (1998) ; Stensvold et al. (2002) ; Stensvold (2008) ; Zika and Farrar (2009) ; Gilman et al.
(2015) .
the alleles for them as fast (F), medium
(M), and slow (S). Genetic identity
(GI) measures between populations
and species and was calculated using
the program POPGENE ( Yeh and
Boyle, 1997 ).
Morphology measurements We made
a number of both quantitative and
qualitative measurements of mor-
phology on the samples from 2007
to 2009 (Appendix S3). We scanned
samples and measured quantitative
traits that include angle and length
measurements of Swartz and Brunsfeld
(2002) using the program ImageJ
( Rasband, 2008 ). We measured  ve
angles on the bottom four pinnae and
calculated 13 length ratios ( Davis,
1983 ) ( Fig. 1 ; Appendix S3). Length
ratios allowed us to quantify the over-
all dimensions of the fern; for exam-
ple, we quantified how broad the
trophophore was in comparison to its
height. We measured rough shape
parameters using circularity (where 1 = perfect circle) of a bottom
pinnae and counted the number of pinnae pairs, for a total of 22
quantitative characters.
Qualitative traits included characters that mirror traditional
species descriptions and keys of Botrychium s.s. (Appendix S3).
To determine morphological distance, we described each qualita-
tive character using binary coding. A Mantel test using the package
vegan ( Oksanen et al., 2015 ) in the program R ( R Core Team,
2014 ) showed that the quantitative and qualitative data sets were
correlated, although at a low level ( r = 0.26, P < 0.001). Given this
correlation, we combined the data sets for subsequent analyses. We
visualized variation in the data using principal component analysis
(PCA) using the program JMP (v. 9, SAS Institute, Cary, North
Carolina, USA) following Zelditch et al. (2004) and Fresnedo-
Ramírez et al. (2011) . To test the sensitivity of the data to analysis
method, we also analyzed morphological data using nonmetric
multidimensional scaling (NMDS) ordination but found no quali-
tative di erences between them and so do not present the latter
Ampli ed fragment length polymorphisms We extracted DNA
from dried 2007–2009 samples using Qiagen DNeasy Plant Mini
kits (Qiagen, Valencia, California, USA). We used a representa-
tive sampling of the individuals used in the morphometric study,
with a focus on B. matricariifolium individuals in addition to its
putative parent species B. angustisegmentum and B. pallidum .
Data from AFLPs were generated from a library of genomic DNA
digested with the restriction enzymes EcoR1 and MseI following
the method of Williams and Waller (2012) . To analyze the DNA
fragments, we used the program GeneMarker v. 1.4 (So Genetics,
State College, Pennsylvania, USA), creating panels and scoring
fragments by hand, using the conservative criteria of Holland et
al. (2008) : intensity levels over 100, band sizes greater than 100,
symmetric peaks, and small bin sizes. To standardize data across
runs, we scored replicates within each run and examined a set of
alleles that ampli ed across all runs.
We translated the data into appropriate  le structures in R
using the source code AFLPdat ( Ehrich, 2006 ). We used NMDS
(following Emshwiller et al., 2009 ) as implemented in vegan
( Oksanen et al., 2015 ) to analyze the data. We examined B. an-
gustisegmentum , B. pallidum , and B. matricariifolium using the
program  v. ( Pritchard et al., 2000 ) to determine
population structure. We followed the procedure of Evanno et
al. (2005) for coding AFLPs and ran simulations of 1,000,000
generations, with 10,000 generations of burn-in and inferred the
distribution of alleles ( λ ). We ran each set with the admixture
model with two runs per k (1–10).  e change in likelihood as
the number of populations ( k ) increases can be more informa-
tive than the likelihood estimate, so we also calculated Δ k
following Evanno et al. (2005) .
Comparing morphology and genetics We performed Mantel
tests with R packages vegan ( Oksanen et al., 2015 ) and ape ( Paradis
et al., 2004 ) to test a possible correlation between morphological
and genetic distances using the Gower distance method ( Gower,
1971 ) for morphological traits and Nei–Li genetic distance
( Nei and Li, 1979 ). We also ran partial Mantel tests controlling
for geographic distances between collection sites using vegan
( Oksanen et al., 2015 ).
Allozymes Of 22 gene loci examined by enzyme electrophoresis,
20 varied within or among members of the B. matricariifolium
complex, their putative diploid parents ( Table 2 ), and related dip-
loid species. At three gene loci in B. matricariifolium and one in B.
michiganense , we found alleles that were not detected in range-wide
sampling of the putative parental species.
We calculated genetic variability parameters of 10% polymor-
phic loci and 1.09 alleles per locus for B. angustisegmentum ,
TABLE 2. Alleles expressed at enzyme-coding loci in B. michiganense and related species. Allele numbers re ect relative migrating positions within genus
Botrychium, with alleles of the presumed diploid parents shaded. Shaded rows indicate loci at which B. angustisegmentum and B. lanceolatum consistently
display di erent alleles. In allotetraploid combinations, the presumed Lanceolatae contribution is listed  rst, using the format of the presumed contributing
diploid, or black where the contributor is ambivalent. Allele numbers in parentheses are orphans that were not detected in any of the three diploid species,
presumed to have been contributed by the non- Lanceolatae parent. The species are B. angustisegmentum (ang), B. lanceolatum (lan), B. matricariifolium (mat), B .
michiganense (mich), B. hesperium (hesp), and B. pallidum (pal). Modi ed from Gilman et al. (2015) and reproduced with permission.
Locus ang lan mat mich hesp pal
Aat-2 3 3 3+( 2 ) 3+( 2 ) or 3+ 3 3+ 3 3
Aat-3 0.2 0.5 0.2 + 2 0.2 + 2 or 0.5 + 2 0.5 + 2 2
Aat-4 0.2 0.5 0.2 + 3 0.2 + 3 or 0.5 + 3 0.5 + 3 3
Aco-2 3 3 3+ 3 3+ 3 3+ 3 2 or 3
Dia-1 1 1 1+ 2 1+ 2 1+ 2 2
Dia-2 1 0.5 1 + 1 1 + 1 or 0.5 + 1 0.5 + 1 1
Dia-3 2.5 or 3 3 3+ n 3+ n 3+ n a N
Dia-4 5 6 5 + 8 5 + 8 n + 8 8
Idh-1 1 1 1+ 1 1+ 1 1+ 1 1
Mdh-1 2 2 2+ 1 2+ 1 2+ 1 1
Mdh-2 3 7 3 + 3 7 + 3 7 + 3 3
Mdh-3 2.5 or 3 2.5 or 3 3+ 2 3+ 2 or 2.5+ 2 3+ 2 2
Mdh-4 2 2 2+ 2 2+ 2 2+ 2 2
6Pgd 4 4 4+ 1 4+ 1 4+ 1 1
Pgi-2 4 4 4+( 1 ) or 4+ 2 or 4 +n or n+ 2 4+ 2 4+ 2 or 4+ n 2
Pgm-1 3 3 3+ 1 or 3+ n 3+ 1 or 3+ n 3+ 1 or n+ 1 1
Pgm-2 1.5 1.5 1.5+ 2 1.5+ 2 1.5+ 2 2
Skdh 2 1 2 + 1 or 2 + n 2 + 1 or 1 + 1 1 + 1 1
Tpi-1 3 3 3+( 1 ) or 3+ 3 3+ 3 3+ 3 3
Tpi-2 3 3 3+ 3 3+ 3 3+ 3 3
a It is assumed that tetraploid plants received two homoeologous copies of each gene, but because these genes are redundant in activity, one of the homoeologous copies may have become
silenced (n). We have not attempted to detect this possibility at loci receiving the same allele from both parental diploids here. Where parental diploids di er in potential contribution but
only one allele is expressed, we suggest that silencing has occurred.
b Diploid Botrychium species regularly express banding patterns re ecting four independently migrating loci for Diaphorase, but B. pallidum expresses only three bands for this enzyme family.
It is possible that Dia-3 has become silenced or that it comigrates with one of the other loci.
TABLE 3. Distribution of B. matricariifolium morphotypes among potential TPI/PGI genotypes in the western Great Lakes region. Leaf silhouettes are representative specimens of the morphotoypes
with the speci ed genotype. Genotypes without silhouettes had no detected pairs of that genotype/morphotype combination. Genotype formulas at the bottom of boxes are the alleles visualized or
expected followed by the number and percentage of the morphotype with that genotype.
PGI genotype
TPI Genotype Morphotype FF– FFSS MM– MMSS SSSS or–SS
No. and %
of morphotype
FSSS acuminate FS/F 0 FS/FS 0
FS/M 21 (38.)
FS/MS 5 (9 .3)
FS/SS 0 26 (48.1)
FS/F 13 (13)
FS/FS 28 (28)
FS/M 0
FS/MS 10 (10)
FS/SS 0 51 (51)
typical FS/F 0 FS/FS 0 FS/M 0 FS/MS 0 FS/SS 0 0
SSSS or–SS acuminate S/F 0 S/FS 0 S/M 0
S/MS 27* (50)
S/S 1 (1.9)
28 (51.9)
S/F 9 (9)
S/FS 35 (35)
S/M 2 (2)
S/MS 3 (3)
S/S 0 49 (49)
S/F 13 (9.4)
S/FS 117 (84.8)
S/M 0
S/MS 5 (3.6)
S/S 3 (2.2)
138 (100)
Total no. and %
of morphotype
acuminate 0 0 21 (38.9) 32 (59.3) 1 (1.9) 54
blocky 22 (22) 63 (63) 2 (2) 13 (13) 0 100
typical 13 (9.4) 117 (84.8) 0 5 (3.6) 3 (2.2) 138
Total 35 (12) 180 (61.6) 23 (7.9) 50 (17.1) 4 (1.4) 292
5% and 1.05 for B. lanceolatum , and 5% and 1.05 for B . pallidum ,
respectively. For the allotetraploid taxa, the respective values were
70% and 1.9 for B. matricariifolium (including morphotypes
“acuminate” and “blocky”), 75% and 1.9 for B. michiganense , and
65% and 1.75 for B. hesperium . We observed allelic variability that
was not attributable to  xed heterozygosity or gene silencing as-
sociated with allopolyploidy in two loci for B. matricariifolium
and six loci for B. michiganense . Considering only these loci, val-
ues for the percentage of polymorphic loci and alleles per locus
were then 10% polymorphic and 1.1 alleles per locus for B. matri-
cariifolium , 30% and 1.05 for B. michiganense , and 5% and 1.05
for B. hesperium .
The degree of genetic similarity among allopolyploid taxa
expressed as genetic identity (GI: Nei, 1972 , 1978 ) was highest
among the morphotypes of B. matricariifolium , with values of
0.995 between “typical” and “acuminate” morphotypes, and 0.954
between “typical” and “blocky.” GI values between B. michiganense
and the morphotypes of B. matricariifolium ranged from 0.807 to
In addition to allelic variability contributed by parental di er-
ences (homoelogous variability) in morphotypes of Botrychi um
matricariifolium , they also displayed allelic variability in contri-
butions from a single parent (homologous variability) at TPI-1
(with two alleles) and PGI-2 (with three alleles). In both cases,
the variable alleles are presumed to be contributed by the B. pal-
lidum parent as the alleles in question have not been detected in
any species of section Lanceolatae . e distribution of morphot-
ypes “typical”, “blocky”, and “acuminate” among potential TPI/
PGI genotypes is shown in Table 3 , with TPI alleles represented
as F (fast) and S (slow) alleles, and PGI alleles represented as F
(fast), M (medium), and S (slow alleles). One half of the TPI/PGI
combinations were undetected in any of the 292 plants sampled
in the WGL region, and no plants of “typical” morphology dis-
played the TPI F allele.  e TPI F allele was expressed in ap-
proximately half of the plants of both “blocky” and “acuminate”
e PGI F allele was expressed in 94.2% of plants with “typical”
morphology, 85% of plants with “blocky” morphology, and in no
plants with “acuminate” morphology.  e PGI M allele was ex-
pressed in 3.6% of “typical” plants, 15% of “blocky” plants, but
98.2% of “acuminate” plants.  e PGI S allele was expressed in 5.8%
of “typical” plants, 13% of “blocky” plants, and 61.2% of “acumi-
nate” plants. Potential origins accounting for the unbalanced distri-
butions of morphotypes are presented in Fig. 5 .
Morphology e ordination of  ve species using 42 morphological
characters showed a pattern in which diploid species were separated,
but with considerable overlap of individuals between species ( Fig. 6 ).
Individuals with missing morphological data were not included.  e
rst three axes in this combined PCA accounted for 25.86% of the
variation ( Table 4 ). e analysis that used only quantitative traits ac-
counted for a higher percentage of the variation in the  rst three axes,
but had a similar pattern to the broader analysis. Characters making
up the  rst three axes included the spacing of the  rst and second
pinnae, broadness of the trophophore, shape of the pinnae, and an-
gles of the second pinnae pair. In general, B. angustisegmentum and
species in section Lunariae species were separated on the perimeter
of the ordination, with allopolyploids B. matricariifolium (including
B. acuminatum and B. michiganense ined.) and B. minganense
(putatively derived from hybridization between B. pallidum and B.
lunaria ) in two broad and overlapping central groups.
AFLPs e NMDS ordination of AFLP data showed discrete
clustering of B. angustisegmentum , B. pallidum , B. neolunaria , and
FIGURE 5 The minimum number of original hybridizations necessary
to produce the genotypes observed in three morphotypes of
Botrychium matricariifolium . Origins 1 and 2 can account for all geno-
types of the “typical” morphotype. Origin 3 alone can account for all
of the acuminate morphotypes. Origin 4 provides the appropriate
genotypes for 96.4% of “typical” individuals. Genotype formulae refer
to TPI/PGI genotypes possessing fast-, medium-, and slow-migrating
FIGURE 6 Principal component analysis of morphology characters in
Botrychium ferns, including 22 quantitative (e.g., angle of  rst pinnae,
trophophore height, and trophophore broadness) and 20 qualitative
characters (i.e., color, surface texture, and trophophore shape). Axes
scores are low, but a pattern with allotetraploid species in between dip-
loid parental species is seen.
B. lunaria ( Fig. 7 ). Allotetraploid individuals formed a di use group
between B. angustisegmentum and B. pallidum . For the allotetra-
ploids of interest ( Fig. 8 ), B. michiganense (numbered 1–5) was dis-
tinct from B. matricariifolium , while B. acuminatum and plants with
“acuminate” and “blocky” morphotypes were in the central cluster
with “typicalB. matricariifolium . One outstanding individual
(number 6) may represent allohexaploid B . pseudopinnatum , which
has a genomic contribution from B. neolunaria ined. Notably, this
individual was displaced toward B. neolunaria in Fig. 7 . We were
unable to relocate this plant or similar individuals in the  eld for
allozyme analysis.
Within pooled species, there is a clear distinction between B. pal-
lidum , B. angustisegmentum , and the allotetrapolyploid group ( k = 3,
Fig. 9A ). Within the allopolyploids,  consistently identi-
ed 10 individuals ( k = 4, Fig. 9B ) that contained more than 0.1 mem-
bership in a di erent genetic group. Individuals of B. michiganense
(numbers 1–5) were separated from B. matricariifolium, as was the
putative species B. pseudopinnatum (number 6). At values of k higher
than three, these individuals were subdivided into two genetic
groups, but no other individual was identi ed so strongly as being
di erent from the main body of B . matricariifolium plants.
Comparing morphology and genetics Mantel tests found a sig-
ni cant correlation between genetic and morphological distance
across all species and within a data set of B. angustisegmentum and
B. pallidum ( Table 5 ). Correlations between genetic and geographic
distance within B. angustisegmentum , B. pallidum , and B. matricari-
ifolium separately were also signi cant. However, there was no cor-
relation between genetic distance and morphology among B.
matricariifolium individuals or between allopolyploids (including
B. michiganense and B. acuminatum ). Partial Mantel tests that con-
trolled for geographic distance while testing the correlation be-
tween genetic and morphological distance were also not signi cant
in the latter cases ( P > 0.07).
Our investigation of cryptic species in the allotetraploid
B. matricariifolium complex found evidence for repeated hy-
bridizations between similar diploid parents that resulted in
morpholo gically similar species and confusing morphotypes within
TABLE 4. PCA components from morphological analyses of Botrychium species. The qualitative data set uses common key and description characters. The
quantitative data set uses angle measurements and length ratios following Swartz and Brunsfeld (2002) , with details in Appendix S3. The combined data set
corresponds to Fig. 6 .
Data set PCA axis one Correlation PCA axis two Correlation PCA axis three Correlation
Qualitative 9.3% of variation 8% of variation 6% of variation
Pinnae margins entire or
−0.29 Pinnules round −0.40 Similar 1st and 2nd
pinnae pairs
Bottom pinna
symmetrical or not
0.32 Terminal trophophore
pinnules rounded
−0.31 3 or more terminal pinnae −0.22
Sporophore branching 0.33 Color −0.27 1st and 2nd pinnae pairs
di erent because
of lobbing
Sporangial thumbing 0.33 Pinnae shape 0.27 1st and 2nd pinnae pairs
di erent because of
Pinnae margins not lobed 0.35 Trophophore ovate/
0.36 1st and 2nd pinnae pairs
di erent because of length
Quantitative 31.9% of variation 17.4% of variation 12.6% of variation
1st pinnae pair to trophophore
end: length of trophophore
−0.36 Trophophore broadness 0.35 Length of pinnae: width
of trophophore
1st pinnae pair: trophophore
−0.36 2nd pinnae whole angle 0.34 Trophophore length 0.38
2nd pinnae pair to trophophore
end: length of trophophore
0.36 Number of pinnae pairs 0.33 Pinnae broadness 0.34
2nd pinnae pair: trophophore
0.37 Trophophore length 0.32 1st pinnae partial angle −0.34
1st pinnae pair: sporophore
0.42 2nd pinnae partial angle 0.56 1st pinnae stem angle 0.29
Both 11.4% of variation 7.98% of variation 6.5% of variation
1st pinnae pair to trophophore
end: length of trophophore
−0.35 1st pinnae whole angle 0.28 Trophophore length 0.30
1st pinnae pair: trophophore
−0.35 2nd pinnae stem angle −0.25 Sporophore length 0.28
2nd pinnae pair to trophophore
end: length of trophophore
0.25 Trophophore broadness 0.25 Pinnae margins −0.25
2nd pinnae pair: trophophore
0.25 2nd pinnae partial angle 0.25 Pinnae shape 0.23
1st pinnae pair: width of
0.32 Number of pinnae pairs 0.24 Deeply dissected 0.23
B . matricariifolium. ese taxa and their origins were clari ed us-
ing genetic evidence derived through enzyme electrophoresis
and AFLP markers.
Cryptic species in the B. matricariifolium complex Morphologi-
cal intermediacy strongly supports the origin of the tetraploid B .
matricariifolium complex by hybridization events between diploid
species in sections Lanceolatae and Lunariae (twice-pinnate and
once-pinnate sections, respectively). Range-wide analysis of all
Botrychium diploids using codominant allozyme alleles (the al-
leles referred to below) points to B. angustisegmentum and/or B.
lanceolatum ( Lanceolatae ) and B. pallidum ( Lunariae ) as the most
likely parents of all members of the B. matricariifolium complex
( Table 2 ). e “best match” for the Lanceolatae parent of tetraploid
B. hesperium from allozyme data is western B. lanceolatum . Lan-
ceolatae parentage of B. michiganense is best explained as having
an origin that predates the eastern–western di erentiation of
these two species because it contains alleles of both B. angustiseg-
mentum and B. lanceolatum ( Gilman et al., 2015 ).  e best match
for the Lanceolatae parent of B . matricariifolium , including mor-
photypes “typical, “blocky”, and “acuminatum” is B. angustiseg-
mentum (see Fig. 2 and Table 2 ).
Within the western Great Lakes (WGL) area where B. michi-
ganense and B. matricariifolium co-occur, we found that each
species had unique alleles. Of 20 loci examined,  ve had alleles
unique to B. michiganense , and two had alleles unique to B . mat-
ricariifolium . ese allelic di erences translate into a genetic
identity (GI: Nei, 1972 , 1978 ) between the two allotetraploids of
0.905, which is high relative to GI numbers typically found be-
tween diploid species ( Crawford, 1985 ; Hamrick and Godt,
1990 , 1996 ). ese high GI values are the result of a shared
B. pallidum parentage common to both tetraploid species.  ese
allotetraploids also share alleles common to both Lanceolatae
parents, but B. michiganense also contains  ve Lanceolatae al-
leles that are now present only in B. lanceolatum and two only in
B. angustisegmentum
. The North American distribution of
B. michiganense is consistent with an origin that pre-dated the
di erentiation of western and eastern Lanceolatae species.
Botrychium michiganense occurs sympatrically with B. lanceola-
tum in the Rocky Mountains from Wyoming northward to southern
British Columbia and Alberta. In boreal regions from northern
Alberta eastward, B. matricariifolium occurs sympatrically with
B. angustisegmentum .
Several observations support recognizing B. michiganense and
B. matricariifolium as distinct species.  ese include the relatively
lower GI between B. michiganense and B. matricariifolium com-
pared with GI values between B. matricariifolium morphotypes, the
seven unique alleles that di erentiate B. michiganense and B. matri-
cariifolium , and likely di erent diploid parentage of B. michi-
ganense and B. matricariifolium .
Allelic di erentiation between the morphotypes within B. mat-
ricariifolium does not match the level of genetic distinction ob-
served between B. matricariifolium and B. michiganense . Although
genotype combinations of TPI and PGI have a level of genotype
correlation with morphotype, these genotypes are neither con-
stant within morphotypes nor limited in most cases to particular
morphotypes. Genetic identity values between the morphotypes
were 0.989 between “typical” and “blocky,” and 0.995 between
“typical” and “acuminate,” the latter morphotype including for-
mally described B. acuminatum . Genetic similarities and mor-
phological gradations observed between the morphotypes support
retaining them all, including B. acuminatum , within the species B.
matricariifolium .
Our multivariate analyses of a mixture of quantitative and quali-
tative traits (see Table 4 ) were only moderately useful in separating
entities within the Botrychium genus. Morphological characters
produced clustering of diploid species, but with overlap between
them, and there was little clustering within the B. matricariifolium
complex ( Fig. 6 ). Instead, these allopolyploids plotted in a di use
array, with individuals arranged across a broad morphospace be-
tween and overlapping with putative parent species.  is pattern
updates our understanding of the range of variation seen in this
group (and the reasons for its frustrating taxonomic issues). It is
important to recognize that this analysis is based on unbiased selec-
tion of characters and includes many characters that the allopoly-
ploid taxa likely share because of intermediacy between two very
di erent parents. Such undiagnostic characters may compromise
the e ectiveness of the relatively few characters that are diagnostic
for taxa across the genus. Separating diagnostic from nondiagnostic
characters is greatly aided by genetic analysis that  rst groups the
same set of plants on the basis of genetic similarity, allowing subse-
quent recognition of character states correlated with speci c groups.
FIGURE 7 Nonmetric multidimensional scaling (NMDS) ordination of
AFLP data for six species of Botrychium in the Great Lakes area, including
two species of the allotetraploid B. matricariifolium complex and their
putative diploid parents. Two diploid species ( B. lunaria and B. neolu-
naria ) and an allotetraploid species ( B. minganense ), all unrelated to the
B. matricariifolium complex are included for scaling purposes. Allotetra-
ploids B. matricariifolium and B. michiganense are positioned between
putative parents B. pallidum and B. angustisegmentum . Allotetraploid B.
minganense is positioned between putative parents B. neolunaria and B.
pallidum ( Farrar, 2011 ). Botrychium lunaria and B. neolunaria occupy dis-
crete areas as proposed by Stensvold (2008) .
Our AFLP data corroborate allozyme results relative to genetic
diversity in the B. matricariifolium complex. Diploid species of
Botrychium clustered together into more discrete groups in genetic
space than in morphological space ( Fig. 7 ). Botrychium matricari-
ifolium, including the “blocky” and “acuminate” morphotypes, and
B. michiganense together form a distinct genetic cluster between
their putative parent species but do not show strong separation
within the group ( Fig. 8 ). Individuals of presumed B . michiganense
are somewhat removed from the central cluster, as is one individual
presumed to be B. pseudopinnatum . Within the tetraploid group,
 identi ed the  ve plants of B. michiganense , but with
only partial distinction from B. matricariifolium , which might be
expected as they both likely share a B. pallidum genome.
e AFLP data also support hypotheses of putative parental spe-
cies for other allotetraploids. In addition to the B. matricariifolium
complex being plotted between Lanceolatae and B. pallidum parents,
the allotetraploid B. minganense places between B. lunaria and B.
pallidum , which are its two putative parents ( Wagner, 1993 ; Hauk
and Hau er, 1999 ).  e outlier of the B. matricariifolium complex,
which we presume to be B . pseudopinnatum , places toward B. neolu-
naria . e genomic constitution of this allohexaploid contains alleles
FIGURE 8 Nonmetric multidimensional scaling (NMDS) ordination of AFLP data for the B. matricariifo-
lium complex. Plants of B. michiganense (numbered 1–5) occupy space peripheral to plants of B. matri-
cariifolium , including morphotypes “blocky” and “acuminate. The outlying plant numbered (6) displays
morphology consistent with that of allohexaploid B. pseudopinnatum , which contains a genome
putatively derived from B. neolunaria ( Farrar, 2011 ). Geographic location (indicated by symbol color)
does not correlate with taxon or morphology. Abbreviations: Co., County; Mtn, Mountain; Nat’l For.,
National Forest.
that are otherwise unique to B. neolu-
naria , and we presume was derived by
hybridization between a diploid mem-
ber of section Lanceolatae and allotet-
raploid B. minganense , the latter of
which contains a genome from B. neo-
lunaria ( Wagner and Wagner, 1990 ;
Farrar, 2011 ). e AFLP separation of
European and North American acces-
sions of B. lunaria ” supports Stens-
vold’s (2008) proposed recognition of a
distinct North American taxon, B. neo-
lunaria Stensvold ined.
Inferred polyploid formation in
Despite the high genetic
and morphological similarity between
B. matricariifolium morphotypes, the
nonrandom distribution of morphot-
ypes among possible TPI/PGI geno-
types ( Table 3 ) requires explanation.
ree alleles of PGI are present through-
out the range of B. matricariifolium ,
but in the WGL study area, the M allele
is universally present in the “acumi-
nate” morphotype.  e TPI F allele
was not detected outside the WGL
area, nor was it detected in any plants
with “typical” morphology within
the WGL area. A high frequency within
the area (it is found in approximately
half of the “blocky” and “typical” mor-
photypes) suggests a local origin of
this allele. It seems unlikely that de
novo mutation within B. matricariifo-
lium producing the TPI F allele would
also contribute the correlated morpho-
logical changes. Conversely, it is more
likely that the new allele was contrib-
uted by a somewhat di erentiated diploid parent. Because this allele
has not been detected in section Lanceolatae and is present in other
species of section Lunariae , B. pallidum is the most likely contributor
of TPI F in B. matricariifolium .
e generation of the genotypes observed in B. matricariifolium
required a minimum of four independent hybridizations between pa-
rental diploids ( Fig. 5 ).  ree of these origins required alleles not cur-
rently present in putative parents B. angustisegmentum or B. pallidum .
ese alleles, TPI F and PGI F, have not been detected in species of
section Lanceolatae in our range-wide sampling, but are present in
other species of section Lunariae . However, these species contain
other alleles and morphologies that are not compatible with them
contributing to the parentage of B. matricariifolium . e most parsi-
monious assumption is that these alleles were therefore at some time
present in B. pallidum , PGI F as a widespread allele and TPI F as a
more local and possibly more recent allele in the WGL area. Based on
these assumptions, we hypothesize the following steps in evolution of
the observed genetic diversity within B. matricariifolium ( Fig. 5 ):
(1) A Botrychium pallidum parent with the TPI/PGI genotype SS/
FF combined in hybridization with a B. angustisegmentum
parent with the genotype SS/SS to produce allotetraploid B.
matricariifolium genotypes SSSS/FFFF and SSSS/FFSS that
are displayed by the most common and widespread “typical
plants of the species, and rare plants with a SSSS/SSSS
(2) e PGI F allele was lost or mutated to current M, and
B. pallidum in the western Great Lakes area that had geno-
types FS/MS combined with B. angustisegmentum SS/SS in
multiple independent hybridizations to produce B. matri-
cariifolium “blocky” and “acuminate” morphotypes with
their multiple allozyme genotypes, and the uncommon
SSSS/MMSS genotype of “typicalB. matricariifolium .
FIGURE 9 Results from STRUCTURE analysis comparing Botrychium species including the Botrychium matri-
cariifolium complex. (A) All individuals ordered by genetic similarity ( k = 3). Numbers correspond to Fig. 8 .
The horizontal line indicates B. michiganense individuals, numbered 1–5. Individual no. 6 is likely the
hexaploid B. pseudopinnatum . (B) Allopolyploids dispersed among geographic populations. Numbers
indicate the same individuals as in (A). Botrychium michiganense individuals are distinct from other indi-
viduals, with high membership in other groups.
TABLE 5. Results of Mantel tests between genetic and morphological distances of Botrychium species. Genetic distance is the Nei–Li genetic distance based on
AFLP data. Morphological distance is the Gower distance based on 42 quantitative and qualitative traits.
Species Distances compared No. individuals No. permutations Correlation P
All Morphological: quantitative vs.
qualitative characters
121 10000 0.262 <0.0001
All Genetic vs. morphology 121 10000 0.350 <0.0001
Botrychium pallidum (2 n ),
B. angustisegmentum (2 n )
Genetic vs. morphology 36 10000 0.648 <0.0001
Botrychium allotetraploids a (4 n ) Genetic vs. morphology 55 10000 0.104 0.108
B. matricariifolium b (4 n ) Genetic vs. morphology 50 10000 −0.074 0.783
Note: Bold text indicates signi cance at P = 0.05 level.
a Includes Botrychium michiganense .
b Includes “blocky” and “acuminate” morphologies.
(3) A er a reduction to its current
SS/MM genotype following the
loss of the TPI F allele, B. palli-
dum may have hybridized with
current B. angustisegmentum
SS/SS to produce the formally
described B. acuminatum , which
is the most localized genotype
of the “acuminate” morpho-
type, described by W. H. Wagner
from a single locality in northern
In considering the possibility of
multiple origins as an explanation for
the morphological variation in allo-
tetraploid B. matricariifolium , we
recognize that the observed morpho-
logical variation within taxa likely
includes genetically based variation
that is not detected in our allozyme
analysis. Significant morphological
variation is often present among
plants of the same size in populations
occupying apparently uniform envi-
ronmental conditions ( Williams and
Waller, 2015 ). Allotetraploids of dif-
ferent morphology can arise through
multiple hybridizations between dif-
ferent morphologies of the same two
parental diploids. Such variation is
displayed in Fig. 10 , with morpho-
logically variable but allozymically
identical plants of B. angustisegmen-
tum and B. pallidum adjacent to B. matricariifolium plants of “typ-
ical”, “blocky”, and “acuminate” morphologies to illustrate how
allotetraploids of di erent morphology might arise through mul-
tiple hybridizations between di erent morphologies of the same
two parental diploids.
Genetic data aid in identi cation of diagnostic morphological
While molecular genetic studies of plant diversity
over the last half-century have largely supported previous con-
cepts based on morphology, some have profoundly altered previ-
ous conclusions.  e latter include groups where morphology was
FIGURE 10 Confusing morphological variation within allotetraploid Botrychium matricariifolium (middle
row) is explained by multiple origins from its diploid parents B. angustisegmentum (top row) and B. palli-
dum (bottom row).
either too di erent from the remainder of ferns ( Rothfels et al.,
2015 ) (e.g., relationships among Equisetales, Psilotales, and
Ophioglossales and between these clades), or too similar (as in
cryptic species) to permit clear morphological distinction of spe-
cies and clades (e.g., cheilanthoid clades recently identi ed
through molecular data, Grusz et al., 2014 ). Revelation of natural
groups using genetic data allows us to identify morphological
characters diagnostic of a particular species or clade that were
previously obscured in a matrix of nondiagnostic variation.  is
is the case in the Botrychium matricariifolium complex, where our
genetic distinction results aide in the recognition of the most di-
agnostic morphological characters ( Fig. 11 ) allowing recognition
of B. michiganense ( Gilman et al., 2015 ). By calling our attention
to unusual genotypes and their correlation with certain morpho-
logical characters in B. matricariifolium , genetic analysis also al-
lowed recognition of genetically based morphological variation
and led to hypotheses of multiple origins of the species by sequen-
tial hybridization events involving the same diploid parents.  us,
genetic analyses can aid  eld botanists by facilitating identi ca-
tion of diagnostic morphological characters and by generating
explanatory hypotheses regarding the evolution of the diversity
that we see.
Dealing with allopolyploids and cryptic species e Botrychium
matricariifolium complex joins the ranks of multiple other taxa
in which independently derived polyploid lineages have been
documented ( Soltis, et al., 1993 , 2014 ; Haufler et al., 1995 ; Beck
et al., 2012 ; Sigel et al., 2014 ). The role of polyploid formation is
increasingly recognized as common and an important process of
diversification and speciation ( Soltis et al., 2014 ). The increase
in allelic variability of allopolyploids
after combination of genomes from
two parental diploids may confer
adaptive  exibility ( Soltis et al., 2004 ).
In B. matricariifolium , observed di-
versity measures are much increased
over those recorded for either pa-
rental diploid (see Table 2 ).  e large
range and frequency of occurrence
of B. matricariifolium also argues for
ecological success of allotetraploid
Systematic studies in taxa that
have multiple independent lineages
of morphologically small simple
plants can be particularly challeng-
ing ( Perrie and Brownsey, 2005 ; Per-
rie et al., 2010b ; Grusz et al., 2014 ).
e combined evidence presented
here supports recognition of a single
and morphologically variable spe-
cies, B. matricariifolium , that in-
cludes B. acuminatum , which was
derived from multiple hybridization
events between the same two ances-
tral diploids. It also is consistent
with recognition of a distinct B.
michiganense species that was de-
rived from genetically di erent an-
cestral diploids that were morphologically similar to those of
B. matricariifolium . While a case could be made for recognizing
“acuminate” and “blocky” morphotypes as varieties of B. matri-
cariifolium , this would introduce signi cant taxonomic prob-
lems. Indeed, for the  eld botanist, the frequent presence of a
continuum of morphologies between such varieties would be
seriously problematic ( Fig. 2 ). Continued studies of allopoly-
ploid origins in Botrychium should concentrate on the patterns
and processes of multiple hybrid origins and how these may cor-
relate with morphology ( Perrie et al., 2010a ; Sigel et al., 2014 ).
Studies on the genetic expression of diagnostic morphological
characters across the genus would also be extremely useful. We
therefore recommend continued recognition of B. matricariifo-
lium , with all its variability, as a single uni ed species that pres-
ents a fascinating window into the origins of allotetraploid
variability in ferns.
e authors thank P. Diggle and two anonymous reviewers for
invaluable comments; D. Waller, D. Baum, C. Ané, and E. Emshwiller
for review of previous dra s; A. Bochte for image analysis assistance;
J. Wendel, J. Nason, and C. Skelton for electrophoresis expertise and
facilities; and L. Loope, B. Leutscher, and S. Trull for help with
permits.  is research was supported by the Clarence R. & Florence
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... Farrar and coworkers were able to circumvent morphological limitations by using applied genetic analyses of 20 enzyme loci to characterize individual taxa and test relationships within the genus (see Farrar 2011 for overview). These studies led to the discovery of additional cryptic species (Farrar and Johnson-Groh 1991;Stensvold et al. 2002;Stensvold and Farrar 2017), and, because enzyme electrophoresis visualizes codominant alleles, it allowed the detection of both parental taxa of allopolyploid species (Zika and Farrar 2009;Gilman et al. 2015;Williams et al. 2016;Meza-Torres et al. 2017). Based on genetic identity (GI) (Nei 1978) between species, DNA sequences (Dauphin et al. 2014), karyological data to determine ploidy level (Wagner 1993;Dauphin et al. 2016), and ecological and relevant morphological characteristics (Farrar 2011), all these authors used the biological species concept to define species and varieties, and we follow their circumscription of taxa. ...
... Initial identification of taxa by genetic analysis of allozymes has also aided morphological identification enabling recognition of certain diagnostic morphological characters Williams et al. 2016 (Gilman et al. 2015;Stensvold and Farrar 2017). This study also evaluates eight unpublished taxa (B. ...
... Angstr. is under study). Second, B. acuminatum W. H. Wagner was not supported as distinct from B. matricariifolium (GI 5 99%) (Williams et al. 2016 , and finally, preliminary analysis of both allozyme and chloroplast data suggest that the species pairs B. mormo W. H. Wagner and B. montanum, and B. dusenii and B. spathulatum have genetic similarity much higher than expected for distinct species and are proposed herein for reevaluation as to proper rank. Some newly recognized clades in this study that may warrant taxonomic recognition, but have yet to be evaluated for GI, are listed as potentially new taxa warranting further study (Table 1). ...
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The moonwort genus, Botrychium s. s., includes diploid and polyploid taxa that occur primarily in the northern hemisphere. Their evolutionary history, morphologically cryptic taxa and deep divergence of the family in the phylogeny of ferns has long fascinated pteridologists. Previous molecular studies did not include a complete taxonomic sampling of the taxa in the genus, nor multiple specimens from throughout the known geographical range of each taxon. Therefore, to investigate evolutionary relationships of the major clades of Botrychium s. s., we increased both taxonomic representativeness (multiple accessions per taxa), as well as phylogenetic resolution by including additional new chloroplast markers. To confirm identification and provide evidence from both maternal and paternal parentage of allopolyploids, we also included specimens that have been characterized by allozyme profiles determined by electrophoretic analysis of 20 nuclear enzyme loci for each taxon. We analyzed four chloroplast regions (matK intron, trnH GUG-psbA, and trnL UAA-trnF GAA intergenic spacers, and rpL16 intron region) of 365 specimens from Asia, Europe, North America, Oceania, and South America, sampling the geographical range of 34 of 35 accepted Botrychium s. s. taxa and thirteen putatively new taxa. We conducted a phylogenetic analysis of maternal lineages based on 2,385 aligned nucleotides using maximum likelihood and Bayesian inference to explore genetic diversity and phylogenetic relationships among taxa. We found strong support for the monophyly of three major clades: Lanceolatum, Lunaria, and Simplex-Campestre, and resolved 15 subclades. Our results suggest multiple origins for at least four polyploid taxa (B. boreale, B. michiganense, B. yaaxudakeit, and B. watertonense). The Simplex-Campestre clade had the largest number of species, despite having a similar total number of haplotypes as the Lunaria clade (62 and 59, respectively), which has the broadest worldwide distribution. In total, our new molecular phylogeny comprises 47 taxa, of which thirteen are discussed for possible taxonomic recognition.
... The relationships within Botrychioideae have been relatively well resolved (Hauk et al., 2012;Dauphin et al., 2014Dauphin et al., , 2016Dauphin et al., , 2017Williams et al., 2016). However, the relationships within Ophioglossoideae remain mysterious or confusing and the taxon sampling needs expansion. ...
... In comparison with the taxon sampling in our most recent study (Zhang et al., 2020a), we added 35 accessions representing 17 species, 12 of which belong to Ophioglossum. Our current taxon sampling is larger mainly in Ophioglossoideae considering that the relationships in Botrychioideae have been well resolved in earlier studies (Hauk et al., 2012;Dauphin et al., 2014Dauphin et al., , 2018Williams et al., 2016). Twelve plastomes were newly generated for this study (Appendix I). ...
Adder's tongue ferns or Ophioglossaceae are best known among evolutionary biologists and botanists for their highest chromosome count of any known organisms, the presence of sporophores, and simple morphology. Previous studies recovered and strongly supported the monophyly of the family and the two multi-generic subfamilies, Botrychioideae and Ophioglossoideae, but the relationships among these and two other subfamilies (Helminthostachyoideae and Mankyuoideae) are not well resolved preventing us from understanding the character evolution. The monophyly of and the relationships in the species-rich genus, Ophioglossum, have not well been understood. In this study, new phylogenetic trees are reconstructed based on four datasets: Sanger sequences of eight plastid markers of 184 accessions, 22 plastomes (12 are new), 29 morphological characters, and combined Sanger and morphological data. Our major results include: (1) the relationships among the four subfamilies are well resolved and strongly supported in Bayesian and parsimony analyses based on plastomes: Mankyua is sister to the rest, followed by Ophioglossoideae which are sister to Helminthostachys + Botrychioideae; (2) Sanger data, plastomes, and combined Sanger and morphological data recovered and strongly supported the monophyly of Ophioglossum in its current circumscription (sensu lato; s.l.) in Bayesian and/or parsimony analyses; (3) within Ophioglossum s.l., four deeply diverged clades are identified and the relationships among the four clades are well resolved; (4) evolution of 34 morphological characters is analyzed in the context of the new phylogeny, among which shape of rhizomes, germination time of spores, shape of early gametophytes, and a number of other characters are found to contain interesting phylogenetic signal; and (5) based on the new phylogeny and character evolution, we propose a new classification of Ophioglossaceae in which the currently circumscribed Ophioglossum is divided into four genera including three new ones: Goswamia, Haukia, and Whittieria considering their molecular, morphological, ecological, and biogeographical distinctiveness.
... Case studies from Isoëtes and Selaginella, both heterosporous groups, demonstrate that frequent hybridization is not linked to a particular gametophyte lifestyle. It is common as well in the moonworts (a Euphyllophyte group resembling Lycopodiaceae in its subterranean gametophyte ecology; Williams et al., 2016;Dauphin et al., 2014Dauphin et al., , 2018. ...
Hybridization occurs often in the genus Diphasiastrum (Lycopodiaceae), which corroborates reports for the two other recognized lycophyte families, Isoëtaceae and Selaginellaceae. Here we investigate the case of D. alpinum and D. sitchense from the Russian Far East (Kamchatka). Their hybrid, D. × takedae, was morphologically recognizable in 16 out of 22 accessions showing molecular signatures of hybridization; the remaining accessions displayed the morphology of either D. alpinum (3) or D. sitchense (3). We sequenced markers for chloroplast microsatellites (cp, 175 accessions from Kamchatka) and for the two nuclear markers RPB and LFY (175 and 152 accessions). A selection of 42 accessions, including all hybrid accessions, was analysed via genotyping by sequencing (GBS). We found multiple, but apparently uniparental hybridization, clearly characterized by a deviating group of haplotypes for D. sitchense and all hybrids. All accessions showing molecular signatures of hybridization in nuclear markers revealed the parental haplotype of D. sitchense, however only the LFY marker differentiated between the parent species. GBS, including 69,819 quality-filtered single nucleotid polymorphisms, unambiguously identified the hybrids and revealed introgression to occur. Most of the hybrids were F1, but three turned out to be backcrosses with D. alpinum (one) and with D. sitchense (two). These observations are in contrast to prior findings on three European species and their intermediates where all three hybrids turned out to be independent F1 crosses without evidence of recent backcrossing. In this study, backcrossing was detected, which indicates a limited fertility of the hybrid taxon D. × takedae. A comparison of accessions of Kamchatkian D. alpinum with plants from Europe indicated possible cryptic speciation. Accessions from the Far East had (i) a lower DNA content (7.0 vs. 7.5 pg/2C), (ii) different prevailing cp haplotypes, and (iii) RPB genotypes, and (iv) a clearly different SNP pattern in GBS. Diphasiastrum sitchense and the similar D. nikoënse, for the latter additional accessions from Japan were investigated, appeared as forms of one diverse species, sharing genotypes in both nuclear markers, although chloroplast haplotypes and DNA content show slight variations.
... We have found evidence for at least six polyploidization events that gave rise to hexaploids. Similar results have been found in other fern complexes in active evolution, with taxa originating from different genetic lineages through multiple hybridization events in different geographical areas showing no difference in frond morphology (Ranker, Floyd & Trapp, 1994;Hunt et al., 2011;Chao et al., 2012;Beck et al., 2012;Williams, Farrar & Henson, 2016). Table S1). ...
The delimitation of lineages in the Cystopteris fragilis complex is complicated by the presence of multiple cytotypes and a lack of defining morphological characters. One character, the production of rugose instead of regular spiny spores, is sometimes associated with a potential Scottish endemic, C. dickieana; however, whether this character is associated with a distinct lineage is uncertain. To better understand the diversity in the C. fragilis complex, we selected 87 C. fragilis samples of known ploidy (4x, 5x, 6x) for sequencing of two plastid loci and we assessed their spore types. These samples represent the variability found in Northern Hemisphere populations, including the type locality of C. dickieana in Scotland. Our analyses revealed two haplotype lineages, which we label the hemifragilis and reevesiana clades, based on their potential relationship to the two presumed diploid parents of C. fragilis. Hexaploids and tetraploids were both polyphyletic. Rugose spores were rarer overall (26% of samples), but five times more prevalent in the hemifragilis clade. Although proper delimitation and understanding of C. fragilis remains a challenge, this study further describes great genotypic and cytotypic complexity present in this complex. Furthermore, rugose-spored plants are widely distributed and should not be associated with a single name.
... Previous population genetic studies based on isozymes have shown a lack of genetic differentiation among morphologically recognized types (Williams et al., 2016), and the low amount of genetic variation detected within Botrychium populations suggests pervasive selffertilization (Farrar, 1998;Hauk & Haufler, 1999;Williams, 2021). ...
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Ferns are the second most diverse group of land plants after angiosperms. Extant species occupy a wide range of habitats and contribute significantly to ecosystem functioning. Despite the importance of ferns, most taxa are poorly covered by genomic resources and within‐species studies based on high‐resolution markers are entirely lacking. The genus Botrychium belongs to the family Ophioglossaceae, which includes species with very large genomes and chromosome numbers (e.g. Ophioglossum reticulatum 2n = 1,520). The genus has a cosmopolitan distribution with 35 species, half of which are polyploids. Here, we establish a transcriptome for Botrychium lunaria (L.) Sw., a diploid species with an extremely large genome of about ~19.0–23.7 Gb. We assembled 25,677 high‐quality transcripts with an average length of 1,333 bp based on deep RNA‐sequencing of a single individual. We sequenced eleven additional transcriptomes of individuals from two populations in Switzerland, including the population of the reference individual. Based on read mapping to reference transcript sequences, we identified 374,463 single nucleotide polymorphisms (SNPs) segregating among individuals for an average density of 14 SNPs per kilobase. We found that all 12 transcriptomes were most likely from diploid individuals. The transcriptome‐wide markers provided unprecedented resolution of the population genetic structure, revealing substantial variation in heterozygosity among individuals. We also constructed a phylogenomic tree of 92 taxa representing all fern orders to ascertain the placement of the genus Botrychium. High‐quality transcriptomic resources and SNP sets constitute powerful population genomic resources to investigate the ecology, and evolution of fern populations.
... Much of what we know about fern population genetics is derived from this method, in part because allozymes were the dominant system used for population genetic studies in the 1980s and 1990s, when the bulk of the research on fern population genetics was conducted ( fig. 2). This method is still used today (e.g., Williams et al. 2016;Stensvold and Farrar 2017) despite its constraints, and several measures of genetic variation discussed above, including %P, Wright's Fstatistics (F and F ST ), and measures of heterozygosity (e.g., H e ), can easily be calculated using allozymes. ...
... About half of all European fern species are polyploid (Reichstein, 1981;Kramer, 1984, Kramer et al., 1995, Tutin et al. 1993, and just under half have arisen, sometimes even repeatedly, from a sterile cross between two (diploid) species (Schneller, 1996;Sessa et al., 2018;Vogel et al., 1999). Multiple origins of fern complexes were often postulated in the literature cited above and have now been demonstrated using molecular techniques (e.g., Soltis et al., 1987;Ranker et al., 1989;Schneller, 1996;Thomson & Alonso-Amelot, 2002;Beck et al., 2012;Sigel et al., 2014;Sigel, 2016;Williams et al., 2016;Yahaya et al., 2016;Dauphin et al., 2017a;2017b); similar evolutionary patterns are known from higher plants (Alix et al., 2017). Hybrids between species belonging to different genera are very rare (Alston, 1940;Reichstein, 1981;Schneller, 1981;Wagner et al., 1992;Rothfels et al. 2015;Engels & Canestraro, 2017;Lehtonen, 2018) and are ascribed to a nothogenus of their own. ...
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In 2014 James J. Georgusis reported that a chance hybrid between Phlebodium aureum ‘Mandaianum’ and Pyrrosia lingua ‘Cristata’ had originated in his greenhouse in Metairie, Louisiana (USA). Though we initially were inclined to consider it a bizarre mutation of one of its putative progenitors, further study of its morphology and its nuclear 2C DNA content revealed it to be intermediate between its putative parents. We thus accept its proposed ancestry and hence describe this plant here in the new nothogenus ×Phlebosia.
... The previous molecular studies paid a lot of attention to Botrychioideae (Hauk et al., 2012;Dauphin et al., 2014Dauphin et al., , 2016Dauphin et al., , 2017Williams et al., 2016), but Ophioglossoideae has remained very much understudied and only up to 12 samples/species of Ophioglossoideae were included in previous studies (Hauk et al., 2003;. ...
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As an ancient lineage of ferns, Ophioglossaceae are evolutionarily among the most fascinating because they have the highest chromosome count of any known organism as well as the presence of sporophores, subterranean gametophytes, eusporangiate sporangia without annuli, and endophytic fungi. Previous studies have produced conflicting results, identifyingsome lineages with unresolved relationships, and have paid much attention to the subfamily Botrychioideae. But the other species‐rich subfamily, Ophioglossoideae, has remained largely understudied and only up to 12 accessions of Ophioglossoideae have been sampled. In this study, DNA sequences of seven plastid markers of 149 accessions (75 in Ophioglossoideae) representing approximately 82 species (approximately 74% of estimated species diversity sensu J. Syst. Evol., 2016, 54, 563) in the family, and two Marattiaceae and two Psilotaceae, are used to infer a phylogeny. Our major results include: (1) Ophioglossaceae are resolved as monophyletic with strong support, and so are all four subfamilies and genera sensu PPG I except Botrypus and Ophioglossum; (2) a new genus Sahashia is segregated from Botrypus so that the monophyly of Botrypus can be retained; (3) the monophyly of Ophioglossum in its current circumscription is uncertain in spite of our large character sampling; (4) there is substantial cryptic speciation in Ophioderma detected by our molecular and morphological study; (5) the recognition of Holubiella is advocated based on its morphology and its sister relationship with Sceptridium; and (6) a novel sister relationship between Botrychium and the JHS clade (Japanobotrychium + (Holubiella + Sceptridium)) is discovered.
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In Memoriam for Don C. Henson, noted botanist and artist from Manistique, Michigan.
The population genetics of ferns, which results from initiation of individuals in a new location (often via long-distance dispersal) plus a wide range of mating systems, merit continued study. In the case of species in the subfamily Botrychioideae (specifically the genera Botrychium and Botrypus), previous work using allozyme and isozyme techniques revealed low genetic diversity and weak population genetic structure. This lack of genetic differentiation between populations is in spite of underground fertilization in the genus resulting in high levels of inbreeding and primarily fixed heterozygosity in tetraploids. In the present study, Amplified Fragment-Length Polymorphisms (AFLPs) were used to examine population genetics and structure of three species in the genus Botrychium and one species in the genus Botrypus. Measures of population genetic diversity were generally low, with the highest measures in the relatively common Botrypus virginianus. Across all species, measures of population differentiation were low and most genetic variation was contained within populations. Bayesian analysis of population structure using the program STRUCTURE corroborated these findings, with inferred genetic clusters that generally did not correspond to geographic collecting locations. These results agree with previous studies, with low genetic diversity within and among populations likely due to self-fertilization that limits outcrossing and long-distance spore dispersal that results in genetically similar populations.
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Reconstructing the origin and evolution of land plants and their algal relatives is a fundamental problem in plant phylogenetics, and is essential for understanding how critical adaptations arose, including the embryo, vascular tissue, seeds, and flowers. Despite advances in molecular systematics, some hypotheses of relationships remain weakly resolved. Inferring deep phylogenies with bouts of rapid diversification can be problematic; however, genome-scale data should significantly increase the number of informative characters for analyses. Recent phylogenomic reconstructions focused on the major divergences of plants have resulted in promising but in- consistent results. One limitation is sparse taxon sampling, likely resulting from the difficulty and cost of data generation. To address this limitation, transcriptome data for 92 streptophyte taxa were generated and analyzed along with 11 published plant genome sequences. Phylogenetic reconstructions were conducted using up to 852 nuclear genes and 1,701,170 aligned sites. Sixty-nine analyses were performed to test the robustness of phylogenetic inferences to permutations of the data matrix or to phylogenetic method, including supermatrix, supertree, and coalescent-based approaches, maximum-likelihood and Bayesian methods, partitioned and unpartitioned analyses, and amino acid versus DNA alignments. Among other results, we find robust support for a sister-group relationship between land plants and one group of streptophyte green algae, the Zygnematophyceae. Strong and robust support for a clade comprising liverworts and mosses is inconsistent with a widely accepted view of early land plant evolution, and suggests that phylogenetic hypotheses used to understand the evolution of fundamental plant traits should be reevaluated.
We describe a model-based clustering method for using multilocus genotype data to infer population structure and assign individuals to populations. We assume a model in which there are K populations (where K may be unknown), each of which is characterized by a set of allele frequencies at each locus. Individuals in the sample are assigned (probabilistically) to populations, or jointly to two or more populations if their genotypes indicate that they are admixed. Our model does not assume a particular mutation process, and it can be applied to most of the commonly used genetic markers, provided that they are not closely linked. Applications of our method include demonstrating the presence of population structure, assigning individuals to populations, studying hybrid zones, and identifying migrants and admixed individuals. We show that the method can produce highly accurate assignments using modest numbers of loci—e.g., seven microsatellite loci in an example using genotype data from an endangered bird species. The software used for this article is available from
Botrychium michiganense W.H. Wagner ex A.V. Gilman, Farrar & Zika is described as a new moonwort species. It is an allotetraploid, most likely descended from hybrids between ancestral B. lanceolatum s.l. and B. pallidum. It has a pinnate to pinnate-pinnatifid trophophore (sterile segment) and is most similar to B. hesperium and B. matricariifolium. These three species can be distinguished by trophophore shape and dissection and B. michiganense is genetically distinct from the others as evidenced by different allozyme profiles. Botrychium michiganense ranges from New Brunswick, the St. Lawrence region of Quebec, the Great Lakes region of the US and Canada, the Black Hills of South Dakota, and west through the Rocky Mountains to eastern British Columbia and Washington.
Two species of Botrychium subgenus Botrychium (moonworts, Ophioglossaceae), Botrychium minganense Victorin and B. crenulatum W. H. Wagner, can sometimes be confused in the field, even by experts, because of their reduced morphology. Botrychium minganense can imitate B. crenulatum, which is more rare. They are afforded different degrees of protection on Federal lands, making the distinctness of these species a question of management, conservation, and systematic interest. The purpose of this study was to compare a morphometric analysis of these two species with an analysis of DNA markers from the same individuals, and to assess their distinctness under each method. Collections were made in Washington, Oregon, Idaho, and Montana from seven populations of B. crenulatum and 18 populations of B. minganense. Each plant was measured, emphasizing characters cited by authors in the original species descriptions. Canonical variate analysis performed on SAS separated the samples into two species groups with 32% overlap. RAPD genetic markers revealed more genetic variation than has previously been documented in moonworts. UPGMA cluster analysis of the similarity of RAPD profiles showed well-defined B. minganense and B. crenulatum clusters, but no distinct clusters within B. minganense that could be correlated with its morphological variability. Small samples of the moonwort species B. lunaria and B. simplex included for comparison also formed distinct clusters. Botrychium crenulatum had seven unique RAPD bands, and identification of B. crenulatum could be confirmed or ruled out with markers from one or two RAPD primers. Both B. crenulatum and B. lunaria have been suggested as possible diploid parents of tetraploid B. minganense. All RAPD markers absent in B. crenulatum but present in B. minganense were also present or polymorphic in B. lunaria, supporting B. lunaria as a possible parent. One very small population of B. minganense showed a monomorphic RAPD profile, consistent with inbreeding, but all other populations had multiple genotypes. Some plants of B. minganense clustered most closely with plants from populations up to 400 km away, suggesting that variation may be introduced into populations by occasional colonization by spores from distant sources.