Diversification and geographical distribution of Psidium (Myrtaceae) species with distinct ploidy levels

Abstract

Key message
Polyploidy (diploid to octoploid) was evidenced from seven Psidium species, besides the outcomes of the whole-genome duplication about the nuclear DNA content, DNA sequence, and distribution.

Abstract
The previous studies have reported the occurrence of polyploid species in Psidium, all deriving from the basic chromosome number x = 11, which is conserved in Myrtaceae. Here, we aimed to assess the ploidy levels of seven Psidium species and to investigate the genomic outcomes of this karyotype change. Data on chromosome number, ploidy level, nuclear DNA content, and DNA sequence (SSR markers) were sought, quantified, and compared to geographical distribution of the studied Psidium species. A euploid series based on x = 11 was evidenced, with diploid, tetraploid, hexaploid, and octoploid species. These species also differed regarding at least one of the other analyzed traits, especially the hexaploids and the octoploid in relation to the others. Diploid species show restricted geographical distribution in the Atlantic Forest, differently from the polyploid species, which occur in several biomes in Brazil. Ploidy level of the Psidium species is related with the nuclear genome size and both seems to be related with species’ geographical distribution. Besides polyploidy, the genetic changes associated with numerical chromosome shift shown in this study, which increases the knowledge about the diversification and distribution of Psidium species.

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https://doi.org/10.1007/s00468-019-01845-2
ORIGINAL ARTICLE
Diversication andgeographical distribution ofPsidium (Myrtaceae)
species withdistinct ploidy levels
AméliaCarlosTuler1,2· TatianaTavaresCarrijo2· ArianeLunaPeixoto1· MárioLuísGarbin2,3·
MarciaFloresdaSilvaFerreira4· CarlosRobertoCarvalho5· MicheliSossaiSpadeto6·
WellingtonRonildoClarindo5
Received: 12 June 2018 / Accepted: 21 March 2019
© Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
Key message Polyploidy (diploid to octoploid) was evidenced from seven Psidium species, besides the outcomes of the
whole-genome duplication about the nuclear DNA content, DNA sequence, and distribution.
Abstract The previous studies have reported the occurrence of polyploid species in Psidium, all deriving from the basic
chromosome number x = 11, which is conserved in Myrtaceae. Here, we aimed to assess the ploidy levels of seven Psidium
species and to investigate the genomic outcomes of this karyotype change. Data on chromosome number, ploidy level,
nuclear DNA content, and DNA sequence (SSR markers) were sought, quantified, and compared to geographical distribu-
tion of the studied Psidium species. A euploid series based on x = 11 was evidenced, with diploid, tetraploid, hexaploid, and
octoploid species. These species also differed regarding at least one of the other analyzed traits, especially the hexaploids
and the octoploid in relation to the others. Diploid species show restricted geographical distribution in the Atlantic Forest,
differently from the polyploid species, which occur in several biomes in Brazil. Ploidy level of the Psidium species is related
with the nuclear genome size and both seems to be related with species’ geographical distribution. Besides polyploidy, the
genetic changes associated with numerical chromosome shift shown in this study, which increases the knowledge about the
diversification and distribution of Psidium species.
Keywords Myrteae· Euploidy· Karyotype· Nuclear DNA content· SSR markers· Guava
Introduction
Polyploidy, euploidy—a numerical chromosome rearrange-
ment characterized by whole-genome duplication (Stebbins
1950; Edger and Pires 2009; Marchant etal. 2016; Spoelhof
etal. 2017)—is arguably the most important karyotype
change that increases the diversification and drive specia-
tion in plants (Edger and Pires 2009; Madlung 2013; Alix
etal. 2017; Slijepcevic 2018). Polyploidy directly leads to
extensive genomic (of the chromosome number to the DNA
sequence), epigenetic, and transcriptomic changes (Dhooghe
Communicated by Alia.
* Wellington Ronildo Clarindo
welbiologo@gmail.com
1 Escola Nacional de Botânica Tropical, Instituto de Pesquisas
Jardim Botânico doRio de Janeiro, RiodeJaneiro,
RJ22460-036, Brazil
2 Laboratório de Botânica, Departamento de Biologia, Centro
de Ciências Agrárias e Engenharias, Universidade Federal
doEspírito Santo, Alegre, ES29500-000, Brazil
3 Programa de Pós-Graduação em Ecologia de Ecossistemas,
Laboratório de Ecologia Vegetal, Universidade Vila Velha,
Rua Comissário José Dantas de Melo, s/n, Boa Vista,
VilaVelha, ES29102-770, Brazil
4 Laboratório de Genética e Melhoramento Vegetal,
Departamento de Agronomia, Centro de Ciências Agrárias e
Engenharias, Universidade Federal doEspírito Santo, Alegre,
ES29500-000, Brazil
5 Laboratório de Citogenética e Citometria, Departamento de
Biologia Geral, Centro de Ciências Biológicas e da Saúde,
Universidade Federal de Viçosa, Viçosa, MG36570-900,
Brazil
6 Laboratório de Citogenética, Centro de Ciências Agrárias e
Engenharias, Universidade Federal doEspírito Santo, Alegre,
ES29500-000, Brazil

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etal. 2011; Marchant etal. 2016; Segraves 2017; Spoel-
hof etal. 2017). Due to these changes, polyploid taxa can
exhibit new phenotypes or even attributes (morphologic,
phenologic, physiologic, and reproductive) in relation to
their counterparts (Levin 2002; Dhooghe etal. 2011; Seg-
raves 2017; Spoelhof etal. 2017; Shu etal. 2018), within
only one or few generations (Otto and Whitton 2000; Beest
etal. 2012).
These novelties potentially influence the ecology (Otto
and Whitton 2000; Soltis and Soltis 2000; Segraves 2017),
as the increased ecological tolerance, allowing the poly-
ploids overlap the niche of their ancestors (Marchant etal.
2016), as well as to colonize new habitats (Stebbins 1985;
Soltis and Soltis 2000; Segraves 2017; Spoelhof etal. 2017).
This hypothesis is supported, among others, by several stud-
ies on polyploid cytotypes of the genera Fragaria (Hancock
and Bringhurst 1981), Eupatorium (Watanabe 1986), Plan-
tago (Van Dijk and Bakx-Schotman 1997), and Aster (Mün-
zbergová 2007). In these studies, diploids are found to have
restricted spatial distributions, contrary to the widely dis-
persed polyploids. Cytogeographical studies have shown that
diploids and polyploids often occupy different regions of
the landscape along ecological gradients, such as moisture,
whereby polyploids are generally capable of occurring in
drier habitats when compared to diploids (Kay 1969; Wata-
nabe 1986; Maherali etal. 2009; Treier etal. 2009).
The polyploidy was estimated to account for the spe-
ciation of 2–4% of today’s flowering plant species, with
woody plants representing a lower fraction (Otto and Whit-
ton 2000). After a century of study (Barker etal. 2016),
the polyploidy has been identified in several taxa, mainly
crops (Alix etal. 2017), being currently considered that the
whole-genome duplication probably occurred in the ances-
tor of all angiosperm plants (Alix etal. 2017; Spoelhof etal.
2017). Nevertheless, the knowledge about the role of this
genomic change in the diversification, speciation, and ecol-
ogy in tropical lineages is still scarce (Husband etal. 2013;
Spoelhof etal. 2017), especially for trees.
One of the polyploidy outcomes is the nuclear DNA con-
tent change (Kron etal. 2007; Slijepcevic 2018), which can
promote modifications in the size and/or number of veg-
etative and/or reproductive structures of a new plant when
compared to its ancestors (Stebbins 1950). These changes
can affect fitness, and include alterations in growth rates,
seed production, the so-called hybrid vigor or heterosis,
coupled with effective dispersal, and higher germination
rates (Baker 1965; Bretagnolle and Lumaret 1995; Otto and
Whitton 2000; Soltis and Soltis 2000; Comai 2005; Sattler
etal. 2016).
In the Angiosperm family Myrtaceae, polyploidy has
mainly been evidenced by chromosome counting in fleshy-
fruited species of the clade Myrtoideae (Andrade and Forni-
Martins 1998; Costa and Forni-Martins 2006a, b, 2007).
This includes the Neotropical Psidium, a monophyletic
group (Lucas etal. 2007; Rivero etal. 2012; Murillo etal.
2012) with rapid diversification rates (Vasconcelos etal.
2017). The genus comprises at least 100 species, distributed
from Mexico and the Caribbean to Argentina and Uruguay
(WCSP 2017). Sixty percent of the Psidium species occur
in Brazil, being found in different biomes, such as evergreen
tropical rain forests (Amazon and Atlantic Forest), savannas
(Cerrado), and semi-arid forests (Caatinga) (BFG 2015). The
large geographical distribution of many Psidium species is
suggested to result from their superior competitive ability
(Soares-Silva and Proença 2006). Staggemeier etal. (2016)
have demonstrated that species of Myrtaceae exhibit a wide
variety of fruit morphology and phenological strategies
that support a variety of frugivorous sizes while retaining
overall ecosystem functionality. Polyploidy can also explain
this success. Psidium species have chromosome numbers of
2n = 22, 33, 44, 55, 66, 77, and 88, deriving from the basic
chromosome number x = 11 (Atchison 1947; Andrade and
Forni-Martins 1998; Bolkhovskikh etal. 1969; Goldblatt
1981; Goldblatt and Johnson 1996; Moore 1977; Costa etal.
2008; Marques etal. 2016). Despite the existence of many
polyploidy events in Psidium, the origin of polyploidy and
its effects on the species’ diversification and geographical
distribution have not been investigated so far. Besides, the
possible relationships among ploidy and geographical ranges
in Psidium may offer a better understanding about how spe-
ciation affects the dispersal and establishment abilities of
tropical species.
The previous studies have suggested the relationships
between polyploidy and different measures of ecological
‘success’ (Stebbins 1947, 1950; Ehrendorfer 1980; Lewis
1980; Thompson and Lumaret 1992; Soltis and Soltis 2000;
Alix etal. 2017; Segraves 2017). However, these hypotheses
have rarely been tested (Segraves 2017) in the tropics, and
the factors that contribute to the success of polyploids have
seldom been identified. The consequences of whole-genome
duplication on species’ geographical distribution remain
marginally explored. Therefore, the main goal of this study
was to expand the knowledge about the Psidium genome,
including the chromosome number, nuclear DNA content,
DNA sequence, and the geographic distribution of six spe-
cies indigenous to Brazil.
Materials andmethods
Sampling
This study included six Psidium species indigenous to Bra-
zil, as well as the naturalized P. guajava L. The selection
was based on differences in the geographical distribution
of the species using the BFG (2015) database. Five of the

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indigenous species occur in two or more Brazilian biomes,
and two are restricted to the Brazilian Atlantic Forest: P.
guineense Sw. is widely distributed across the different Bra-
zilian biomes (except the Pampas); P. myrtoides O. Berg
and P. cattleyanum Sabine occur in the Cerrado, Caatinga
and Atlantic Forest; P. longipetiolatum D. Legrand is found
in the Cerrado and Atlantic Forest; and P. oblongatum O.
Berg and P. cauliflorum Landrum & Sobral are restricted
to the Atlantic Forest. Young and healthy leaves, from five
individuals for each species, were collected in field expedi-
tions and stored in silica gel for molecular analysis. As the
number of fruits varied between the Psidium individuals,
all fruits were collected for flow cytometry and cytogenetic
analyses, being: 29 for P. guajava, nine for P. oblongatum,
six for P. cauliflorum, 55 for P. guineense, 50 for P. catt-
leyanum, 38 for P. myrtoides, and 18 for P. longipetiolatum.
One voucher per population was collected, dried (Peixoto
and Maia 2013), and deposited at the RB herbarium of the
Botanical Garden of Rio de Janeiro: P. guajava (Tuler, A
445), P. oblongatum (Carrijo, T 2105), P. cauliflorum (Tuler,
A 511), P. guineense (Tuler, A 487), P. cattleyanum (Tuler,
A 427), P. myrtoides (Tuler, A 451), and P. longipetiolatum
(Tuler, A 450).
In vitro establishment, nuclear 2C value
measurement, andchromosome number
determination
Seeds of the seven Psidium species and Solanum lycoper-
sicum Mill ‘Stupické’ (reference standard, 2C = 2.00pg;
Praça-Fontes etal. 2011) were disinfested under laminar
flow hood (Oliveira etal. 2013) and inoculated into flasks
containing MS medium (Murashige and Skoog 1962) sup-
plemented with 3.0% (w/v) sucrose and 0.7% (w/v) type A
agar, pH 5.7. The flasks were maintained at 25°C under a
16/8h light/dark regimen, with 36µmol m−2 s−1 light radia-
tion provided by two fluorescent lamps (20W, Osram®).
As performed by Marques etal. (2016), from the invitro
plantlets, leaves were collected for 2C value measurement,
and roots were collected for 2n chromosome number deter-
mination. The use of invitro plantlets was important owing
to the unavailability of fruits during all months of the year
in which the study was executed.
Nuclear 2C value measurement by flow cytometry is rel-
evant to screen the polyploid taxa and record ploidy changes,
as well as the increase and decrease in genome size that
occurs after this event (Bennetzen and Kellogg 1997; Petrov
2002; Soltis etal. 2003). Therefore, leaf fragments of S.
lycopersicum ‘Stupické’ plantlets (reference standard) grown
invitro and of each Psidium species (samples) were chopped
together, and the nuclei were extracted and isolated (Otto
1990; Coser etal. 2012; Marques etal. 2016). The resulting
suspensions were stained with buffer containing propidium
iodide (Praça-Fontes etal. 2011; Coser etal. 2012) and
analyzed in a Partec PAS® flow cytometer (Partec® GmbH,
Munster, Germany) (Coser etal. 2012; Marques etal. 2016).
The FlowMax® software (Partec®) was used to analyze the
histograms. The mean nuclear genome size (2C) was meas-
ured by dividing the mean channel of the fluorescence peak
corresponding to the standard’s G0/G1 nuclei by that of each
sample. At least 20 invitro plantlets were used for each
species.
Just as for flow cytometry, invitro plantlets were funda-
mental to accomplish the cytogenetic evaluation. From these
plantlets, roots were removed and immediately treated with
4µM amiprophos-methyl (APM, Nihon Bayer Agrochem K.
K.®) for 5h at 30°C. The roots were washed with distilled
water (dH2O) for 20min, fixed in fresh methanol:acetic
acid (Merck®) solution (3:1), stored at -20°C for at least
24h, washed again with dH2O for 20min, and macer-
ated with enzymatic solution for 2h at 34°C (Coser etal.
2012; Marques etal. 2016). Root meristem dissociation
and air-drying (Carvalho etal. 2007) procedures were used
to prepare the slides, which were analyzed using a Nikon
80i microscope (Nikon, Japan). Metaphase images were
captured with a Media Cybernetics® Evolution™ charge-
coupled device (CCD) video camera (Nikon, Japan) coupled
to this microscope.
Molecular analysis
The transferability rates of 141 SSR (simple sequence
repeat) markers were obtained from a previous study (Tuler
etal. 2015). Of these, 32 SSR were selected which ampli-
fied for the seven species analyzed in this study (Table1).
Details of DNA extraction and SSR amplification are avail-
able in Tuler etal. (2015). The number of alleles per locus
and observed heterozygosis per primer was estimated. Data
obtained for alleles of each individual were subjected to dis-
similarity index analysis using the weighted index.
Results
In vitro establishment, nuclear 2C value
measurement, andchromosome number
determination
All seeds of Psidium and S. lycopersicum germinated
after 30 days, providing morphologically normal plant-
lets, which were maintained under controlled environmen-
tal invitro conditions. From the invitro plantlets, nuclear
genome size analysis evidenced high intrageneric varia-
tion of mean 2C values among the seven Psidium species.
Psidium cauliflorum showed the lowest nuclear genome
size, 2C = 0.93 ± 0.002pg (1C = 0.465pg), followed by P.

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guajava, 2C = 0.95 ± 0.021pg (1C = 0.475pg) and P. oblon-
gatum, 2C = 0.98 ± 0.004pg (1C = 0.490pg). In comparison,
the other species presented significantly greater mean values:
P. guineense exhibited 2C = 1.86 ± 0.003pg (1C = 0.930pg),
a nuclear genome size approximately twofold higher than
in P. cauliflorum, P. guajava, and P. oblongatum. P. myr-
toides showed 2C = 3.07 ± 0.0045pg (1C = 1.535 pg), or
3.30 times higher; P. cattleyanum had 2C = 3.57 ± 0.00pg
(1C = 1.785pg), or 3.84 times higher; and P. longipetio-
latum displayed 2C = 5.12 ± 0.002pg (1C = 2.560pg), or
5.51 times higher than in the three above-mentioned species.
Thus, mean 2C value data suggest that karyotype alterations
occurred during evolution, promoting a strong variation in
nuclear genome size (Fig.1; Table2).
Based on this hypothesis, a cytogenetic approach was
performed from the same invitro plantlets to assess the
chromosome number of each species, as well as polyploidy
in P. guineense, P. myrtoides, P. cattleyanum, and P. longi-
petiolatum. Psidium cauliflorum, P. guajava and P. oblonga-
tum presented 2n = 22 chromosomes; P. guineense 2n = 44;
P. myrtoides and P. cattleyanum 2n = 66; and P. longipeti-
olatum 2n = 88. Therefore, cytogenetics revealed a euploid
series comprising diploid (P. cauliflorum, P. guajava, and
P. oblongatum), tetraploid (P. guineense), hexaploid (P.
myrtoides and P. cattleyanum), and octoploid species (P.
longipetiolatum) (Fig.1; Table2).
Considering the non-replicated monoploid genome
x = 11 for the Psidium species sampled here, the 1Cx DNA
Table 1 SSR primers used in the detection of polymorphism amongthe seven species of Psidium
FR: functional region = yes (1), no (0), absent (–)
SSR primer Forward Reverse FR Number
of alleles
mPgCIR 2 AGT GAA CGA CTG AAG ACC TTA CAC ATT CAG CCA CTT 0 5
mPgCIR 16 AAT ACC AGC AAC ACCAA CAT CCG TCT CTA AAC CTC 0 4
mPgCIR 19 AAA ATC CTG AAG ACG AAC TAT CAG AGG CTT GCA TTA 0 4
mPgCIR 21 TGC CCT TCT AAG TAT AAC AG AGC TAC AAA CCT TCC TAA A 0 3
mPgCIR 26 CTA CCA AGG AGA TAG CAA G GAA ATG GAG ACT TTG GAG 0 8
mPgCIR 91 GCG GTG GAT TTG AAT TTA G CCA AGT AAC CCA CAA CAA TA 1 6
mPgCIR 94 CAA CCT TCC CGT GAT TAT T CTA GCT TCT TCA GTG GGA AC 1 9
mPgCIR 97 GAC CTC AGT AGT TCA GCA TGT TAG AGT GGA CGG GAG GAG 1 4
mPgCIR 98 CAT CAA CTT TCC AGG CAT A CCA TTC AGT CGG TTT GAC 1 5
mPgCIR 99 TCA AAG TCC AAA ACT CAT GC GGG ATG GAG TAA AGA TGA AA 1 8
mPgCIR 104 ATT CCC GTG GAT TAT GTA TC ACA ACC ATT TTC TCC TCA TC 1 5
mPgCIR 108 AGG ACC TCA CAG AAG TTC AC CGC TGT TTA CAC TGT CGT T 0 5
mPgCIR 137 GGG GAA TGC AGA GAT TGT AGA TGA TGG TCT CGC TTT T 1 5
mPgCIR 148 CAT ACA GAG TCG GAT GGT TT GCT GCT GGT CTT AAA GCT AA 1 9
mPgCIR 158 ATC ACC ACT ACT CCA CTC GT TAG AAG GTG CTC TAG GCT CA 0 1
mPgCIR 163 TCT TTG CAC ATC AAA CTC G CAT GGT ATC AAT AGG TCA AGC 1 3
mPgCIR 188 TGG ATG AAT CAG GAG GAT TA TTG TGG GGA AGA AAC TAC TG 1 2
mPgCIR 192 ACG CTA ACT ATC GAA ATG CT ACT ACG CAC TTG ATG GAG AT 1 6
mPgCIR 198 CTC GAT CAG AAG AAC AAC ATC ACT GTT CCT GAT GGC TCT C 1 7
mPgCIR 209 CTA AAG CCA CAT CCA GCA CTA ACA TTT GCC TTC TAC AGC 1 4
mPgCIR 231 CTC CAA GAA AAT GGA AAG G TGA AAA CAC CAA ACA GCA C 1 3
mPgCIR 233 GAC TGA AGA CCC AAA TAC CA TTA GGC TGA AAT GCT CCT TA 1 2
mPgCIR 242 TTA AGG TGG GAC CAA GAA G GAC GTA TCG GAT CAA GTT TC 1 4
mPgCIR 256 AGG TGC ATG ATT ACG ATT CT CGA GGT TCT TGA TGT TGT CT 1 6
mPgCIR 277 AGC CGA TTA TGA TTA CCT GA CGA TTC ACT CCC TCA TTA CT – 6
mPgCIR 287 GCT GGT GCA AAA GTA GTC A GCA GTT CTT TTC CTT CTA ACC – 4
mPgCIR 345 CTG GGA GAC TTT TCA AGG GAG TCC GAT GTT GAT GAA G – 5
mPgCIR 347 CTC TGA AAG GGA GAG GAC TT AGA ATC TTC GCC TAT TGC TT – 4
mPgCIR 414 AAC AAC ACG CTT TGA AGT TT CCC AGA AAG ATG AGA CAA AG – 4
mPgCIR 420 CAA CTT TGC TAG AGA TGA AGC ATG TAG TAA TCG AAG AAA TGGTT – 5
mPgCIR 437 ACA ACA GTT CTG ATC CCA AA CTC GGA GAC ACA GAG GTC TA – 3
mPgCIR 439 GCA TCT TGC TTC TGT CAC TT GGA GAT GTG CAA CGT ATT TT – 3

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value (DNA content of basic chromosome number x; Greil-
huber etal. 2005) was: 0.465pg for P. cauliflorum and P.
guineense; 0.475pg for P. guajava; 0.490pg for P. oblonga-
tum; 0.512pg for P. myrtoides; 0.595pg for P. cattleyanum;
and 0.640pg for P. longipetiolatum.
The diploid species, P. cauliflorum and P. oblongatum,
are endemic to the Atlantic Forest, restricted to a few loca-
tions, mainly in rainforest regions. The octoploid P. longi-
petiolatum is also restricted to the Atlantic Forest, occur-
ring in Ombrophilous Forest, Semideciduous forest in the
states of southeastern (Espírito Santo, Minas Gerais, Rio
de Janeiro, and São Paulo states) and Mixed Ombrophil-
ous Forest in south (Paraná, Rio Grande do Sul, and Santa
Catarina states). The tetraploid (P. guineense) and hexaploid
(P. myrtoides and P. cattleyanum) are widely distributed in
Brazil, occurring under different environmental conditions
in the Atlantic Forest, Caatinga, Cerrado, and Amazon Rain-
forest (Fig.2).
Fig. 1 Schematic histogram and karyotype of the seven Psidium
species. Flow cytometry was executed separately for each spe-
cies using the internal standard S. lycopersicum (2C = 2.00 pg;
Praça-Fontes etal. 2011). G0/G1 nuclei peaks of each Psidium spe-
cies are represented in the same histogram, as follows: P. cauliflo-
rum (2C = 0.93 pg), P. guajava (2C = 0.95 pg) and P. oblongatum
(2C = 0.98 pg) in channel 100; P. guineense (2C = 1.86pg) in chan-
nel 200; P. myrtoides (2C = 3.07 pg) in channel 323; P. cattleyanum
(2C = 3.57 pg) in channel 376; and P. longipetiolatum (2C = 5.12pg)
in channel 539. Following the lines from each G0/G1 peak, the karyo-
type of each species is shown: a P. cauliflorum, b P. guajava and c
P. oblongatum with 2n = 2x = 22 chromosomes; d P. guineense with
2n = 4x = 44 chromosomes; e P. myrtoides and f P. cattleyanum with
2n = 6x = 66 chromosomes; and g P. longipetiolatum with 2n = 8x = 88
chromosomes. Note the euploid series in these Psidium species based
on x = 11, the similar nuclear genome size of the diploid species, and
the clear nuclear DNA content difference between hexaploid species.
Bars = 5µm
Table 2 C: 2n chromosome number (ploidy level), D: mean 2C
nuclear DNA content (pg), T: transferability (data from Tuler et al.
2015), H: percentage of heterozygous loci for 32 SSR markers, A:
mean of the alleles in seven species of Psidium
Species C D T H (%) A
Psidium guajava 22 (2X) 0.95 100 34.3 1.40
Psidium oblongatum 22 (2X) 0.98 80.9 37.5 1.59
Psidium cauliflorum 22 (2X) 0.93 53.9 0.00 1.00
Psidium guineense 44 (4X) 1.86 97.8 40.0 1.68
Psidium cattleyanum 66 (6X) 3.57 74.4 34.3 1.43
Psidium myrtoides 66 (6X) 3.07 80.9 37.5 1.50
Psidium longipetiolatum 88 (8X) 5.12 65.2 50.0 1.81

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Molecular analysis
Thirty-two SSR markers were chosen amongst the 132 SSR
markers developed for P. guajava, according to Tuler etal.
(2015), based on transferability in the six Psidium species
(Table2). Diploid species showed the lowest heterozygo-
sity rates, with P. cauliflorum (2x = 22) having all 32 loci in
homozygosis (heterozygosity rate of 0.00%), correspond-
ing to a mean allele number per locus equivalent to 1.00.
In contrast, the octoploid P. longipetiolatum exhibited the
highest heterozygosity rate (50.00%), as well as the highest
mean number of alleles per locus among all species (1.81)
(Table2).
The 32 SSR loci differed among the species. A total of
149 alleles were amplified, with a mean of 4.6 alleles per
locus. The SSR loci mPgCIR 26, mPgCIR 94, mPgCIR 99,
and mPgCIR 148 produced the largest number of alleles (8
or 9), whereas mPgCIR 158, mPgCIR 188, and mPgCIR
233 generated the smallest (1 or 2). In general, SSR from
transcribed regions showed more allelic forms (5.5 alleles
per locus) than those from non-transcribed regions (4.8
alleles per locus) (Table1).
Fig. 2 Distribution of the seven Psidium species in Brazilian biomes
based on data from the literature, herbaria, and the present study.
Distribution of the diploid species (2n = 2x = 22 chromosomes): P.
cauliflorum (blue circle), P. guajava (black circle), and P. oblon-
gatum (yellow circle). Note that P. cauliflorum and P. oblongatum
(yellow circle) only occur in the Atlantic Forest. Tetraploid species
(2n = 4x = 44 chromosomes): distribution of P. guineense (black
square) was not registered in Pantanal and the Pampas. Hexaploid
species (2n = 6x = 66 chromosomes): P. cattleyanum (green triangle)
and P. myrtoides (red triangle) occur in the Atlantic Forest, Cerrado,
Caatinga, and the Pampas. Octoploid species (2n = 8x = 88 chromo-
somes): P. longipetiolatum (blue hexagon) is found distributed in the
Atlantic Forest from Minas Gerais to Rio Grande do Sul. The coor-
dinates used to define the species’ geographical distribution were
obtained with the application Google Earth, using the locations
indicated on the labels of the herbarium specimens. The map of geo-
graphical distribution was made using the program DIVA GIS 5.4

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Discussion
Nuclear 2C value, chromosome number, and molecular
data evidenced the euploid, and dynamic and progres-
sive genomic modifications in the seven Psidium species,
expanding the data about tropical tree species (Husband
etal. 2013; Spoelhof etal. 2017). The four polyploid spe-
cies of Psidium in this study are an example of natural
euploidy derived from whole-genome duplication. There-
fore, these species were originated from intraspecific
whole-genome duplication (autopolyploidy—endorep-
lication or endomitosis of the zygote or fusion of non-
reduced reproductive cells), or from the interspecific
crossing (allopolyploidy—hybridization) involving or
not the whole-genome duplication (Stebbins 1950; Sattler
etal. 2016; Shu etal. 2018). This is a dramatic quest that
remains open, which must be look for each species. Due
to the relatively recent (~ 9.9–20.8Ma, Oligocene–Mio-
cene) radiation of the tribe Myrteae (Thornhill etal. 2015;
Berger etal. 2016), these Psidium species represent recent
polyploids.
The progressive increase in nuclear DNA content,
from the diploid species P. cauliflorum, P. guajava, and
P. oblongatum to the polyploid species P. guineense, P.
cattleyanum, P. myrtoides, and P. longipetiolatum, indi-
cates strong karyotype differences related to numeri-
cal changes (euploidy). Therefore, there is a relation
between the chromosome number and the nuclear genome
size of the seven species. Interspecific and intraspecific
variations in nuclear 2C value in Psidium have been
reported for P. guajava ‘White’—2C = 0.507pg and P.
guajava ‘Red’—2C = 0.551pg (Costa et al. 2008), P.
acutangulum—2C = 1.167pg (Costa etal. 2008), P. aus-
trale—2C = 2.97pg (Souza etal. 2015), P. guineense—
2C = 1.85 pg (Marques et al. 2016) and 2C = 2.02 pg
(Souza etal. 2015), and P. cattleyanum—2C = 1.053pg
(Costa etal. 2008) and 2C = 1.99–5.47pg (Souza etal.
2015).
Chromosome counting corroborated the obtained
2C values and shed light on the karyotype divergences
(Fig.1). Diploidy was confirmed for P. guajava (Costa
etal. 2008; Souza etal. 2015; Marques etal. 2016), as well
as tetraploidy for P. guineense (Souza etal. 2015; Marques
etal. 2016) and hexaploidy for P. cattleyanum (Souza etal.
2015). Nuclear genome size and chromosome number
were characterized for the first time in P. cauliflorum and
P. oblongatum, which exhibited the same nuclear 2C value
and chromosome number as P. guajava. The family Myrta-
ceae is basically diploid (2n = 2x = 22), as illustrated by
Australasian species of the genera Eucalyptus and Mela-
leuca (Atchison 1947; Brighton and Ferguson 1976; Rye
1979). The tribe Myrteae also displays a predominance of
2n = 2x = 22, except for Eugenia, Myrcia, and Psidium, in
which polyploid species are also found (Costa and Forni-
Martins 2006a, b, 2007; Silveira etal. 2017).
Considering polyploid species, P. myrtoides shows the
same 2n = 6x = 66 chromosomes as P. cattleyanum, but its
2C value is 0.50pg lower than in the latter. These results
suggest the occurrence of structural chromosome changes
during the karyotype evolution in Psidium. Besides
euploidy, the karyotype evolution also involves aneuploidy,
which did not observed in the Psidium species of this study,
and structural chromosome rearrangements (Sattler etal.
2016; Slijepcevic 2018). Alternatively, P. cattleyanum and
P. myrtoides may have originated from distinct progenitors
(auto- or allopolyploids). The number of 2n = 8x = 88 chro-
mosomes reported here for P. longipetiolatum has been pre-
viously reported for P. cattleyanum (Atchison 1947), rein-
forcing that polyploidy occurs in this genus. Contrary to the
previous studies (Atchison 1947; Costa and Forni-Martins
2006a, b; Costa etal. 2008; Souza etal. 2015), no variation
in 2C value or chromosome number was found between the
distinct individuals of P. guajava and P. cattleyanum.
The interspecific differences in the nuclear DNA content
among the Psidium species (Fig.1) represent outcomes of
the polyploid origin due that the basic chromosome number
x = 11, which is conserved in Myrtaceae. A polyploid series
from x = 11 was confirmed for Psidium, as well as the mean
nuclear 2C value was showed for each ploidy level (Fig.1).
The polyploidy has mainly been reported for the tribe Myr-
teae (Silveira etal. 2017; Costa and Forni-Martins 2006a, b,
2007), which includes fleshy-fruited species in South Amer-
ica. According to the phylogenetic relationships proposed
for Myrtaceae (Vasconcelos etal. 2017), the tribe Eucalyp-
teae is a basal clade in the subfamily Myrtoideae. The dip-
loid species of the tribes Eucalypteae present 2C = 1.13pg
(Eucalyptus globulus, Azmi etal. 1997). Differently, for the
Psidium sampled here, the mean value for nuclear genome
size (2C = 1.85pg for P. guineense to 2C = to 2C = 5.75pg
for P. longipetiolatum) increased through polyploidy events.
The SSR markers showed that polyploidy in Psidium also
resulted in higher heterozygosity rate and mean number of
alleles. This is a direct effect of this karyotype change. The
high polymorphism of the primers mPgCIR 26, 94, 99, and
148 was a result of the higher number of alleles present
in polyploid species (P. cattleyanum: 3 alleles for mPgCIR
94; P. myrtoides: 4 alleles for mPgCIR 26; P. guineense: 4
alleles for mPgCIR 148; and P. longipetiolatum: 4 alleles for
mPgCIR 99 and 4 alleles for mPgCIR 94). The occurrence of
more than two alleles per SSR locus has also been reported
in accessions of P. guajava (mPgCIR 253, Aranguren etal.
2010), P. guineense, P. cattleyanum, and P. friedrichsthali-
anum (mPgCIR 255, Costa and Santos 2013).
Most of the polymorphic primers are derived from func-
tional regions (Table1; e.g., mPgCIR 94, 99, and 148).

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1 3
Functional regions are associated with control and varia-
tion of adaptive characteristics and/or important traits for
occupation of new habitats, thus affecting the species’ dis-
tribution (Grattapaglia etal. 2012). Based on nuclear 2C
value, chromosome number, and molecular data, we suggest
that the large ecological and geographical amplitudes that
the four polyploid Psidium species occupy can be linked to
their polyploid condition.
The polyploid Psidium species (P. guineense, P. cattleya-
num, P. myrtoides, and P. longipetiolatum) have large geo-
graphical distribution compared to the endemic and diploid
species (P. cauliflorum and P. oblongatum). For instance, P.
guineense occurs in all Brazilian biomes (Atlantic Forest,
Caatinga, Cerrado, and Amazon Rainforest), and this broad
geographical distribution comprises a wide range of envi-
ronmental conditions. Psidium guajava is the only diploid
species presenting large geographical distribution in Brazil.
This can be explained by its cultivation for economic pur-
poses. Considering that polyploids show broader ecological
tolerances and higher colonization abilities in comparison
to diploids (Stebbins 1950; Grant 1981), it is possible that
the phenotypical diversity presented by the four evaluated
polyploid Psidium species enables their exploration of new
habitats.
Conclusion
Euploidy, based on the basic chromosome set x = 11, was
confirmed for four of the seven Psidium species studied here.
The chromosome number explains the increase in nuclear
genome size and genetic diversity, discriminating the tetra-
ploid and octoploid species. Therefore, polyploidy contrib-
uted to the diversification in the studied Psidium species,
representing an important mechanism of speciation. As a
challenging field, further understanding of the evolutionary
history and diversification of Psidium will probably require
approaches including the understanding of the phenotypic
variation associated with the species’ geographic distribu-
tions, and the development of phylogenetic studies. Such
studies, however, will be better understood in the light of
the cytogenetic and molecular patterns revealed in this study.
Author contribution statement ACT, TTC, MLG, and WRC
conceived, designed, and conducted the study. ACT, ALP,
and TTC identified the species of Psidium. ACT, MSS, and
WRC carried out the cytogenetic analyses. WRC and CRC
performed the flow cytometry analysis. ACT and MFSF
conceived and conducted the molecular marker analysis.
All authors equally contributed to the writing, editing and
revision of the manuscript, and approved the final version
for submission.
Acknowledgements We would like to thank the Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq, Brasília—DF,
Brazil; Grants 443801/2014-2 and 308828/2015-1, 305821/2016-4),
Fundação de Amparo à Pesquisa do Espírito Santo (FAPES/VALE,
Vitória—ES, Brazil; Grant 75516586/16), Fundação Carlos Chagas
Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, Rio
de Janeiro—RJ, Brazil) and VALE for financial support. This study was
financid in part by the Coordernação de Aperfeiçoamento de Pessoal
de Nível Superior—Brasil (CAPES)—Finance Code 001.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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