2. Method
2.1. Study area
The study area is limited to the upper area of the Papaloapan River in
the state of Oaxaca, southern Mexico. In this area the rivers drain from
a maximum altitude of 3,216 m a.s.l. towards a minimum altitude of 1,833
m a.s.l. Strahler order of rivers ranges from 1 at sites in the smallest
rivers (headwaters) to 5 at sites that are located on the largest rivers
(downstream). The flow ranges from 0.01 l/s to 2.5 l/s and has a
negative relationship with altitude (Flores-Galicia et al., 2021). The
climate in the upper part of the watershed corresponds to humid
temperate with summer rains and in the lower and middle part it
corresponds mainly to sub-humid temperate with summer rains. The upland
forests are Abies hickelii and Pinus hartwegii forests in
the upper areas and mixed forests of A. hickelli -Pinus
patula and Pinus pseudostrobus var.apulcensis -Quercus crassifolia towards the middle and low
areas of the watershed (Piña & Trejo, 2014). The vegetation of the
riparian zone changes along the streams, in the headwaters arboreal
elements of species such as A . hickelli , Pinusspp., Quercus spp., towards the intermediate parts Alnus
acuminata , Oreopanax xalapensis and Meliosma dentata ,
while in the lower areas A . acuminata and Salixhumboldtiana are observed (Flores-Galicia et al., 2021).
To evaluate the changes in the communities of propagules, nine sampling
sites were established in two tributary rivers (Yavesía’s river and
Lachatao’s river) that are part of the same drainage network in the
basin, the location of the sites was determined based on a progressive
criterion of distance with respect to the origin of the rivers (Fig. 1).
2.2. Sampling propagules
To characterize the communities of propagules dispersed by hydrochory
and other types of dispersal in the riparian zone, generative propagules
were collected, that is, those that are the product of the sexual
reproduction of plants such as seeds or fruits (Pijl, 1969) in two types
of traps that were placed in sediment deposition zones adjacent to the
streams: (i) Leyer boxes to collect propagules dispersed by hydrochory
and (ii) mesh traps to collect propagules dispersed by other types of
dispersal. Leyer boxes (sensu Leyer, 2006) were boxes open at the
bottom, covered by a 0.15 mm mesh size at the top and fastened by wires.
The construction ensured the free flow of water, sediments, and
propagules into the box, inside an artificial turf mat were placed
(0.5x0.5 m) to retain the propagules. The boxes were tied to 75 cm long
metal rods to prevent them from being dragged by the waterflood (Araujo
et al., 2015). On the other hand, the mesh traps to collect propagules
dispersed by other types of dispersal consisted of extended squares (1.5
× 1.5 m) of shade cloth (1.5 mm mesh) that were placed near the Leyer
boxes. Leyer boxes were placed at the lowest line of perennial
vegetation (greenline) (Fig. 1c).
In each of the nine sampling sites, two Leyer boxes and three mesh traps
were placed to collect propagules dispersed by hydrochory and by other
types of dispersal (Fig. 1b). The traps were visited every two or three
months to collect the propagules trapped in them between January 2017
and January 2018 (they were visited five times: March, June, August, and
November 2017 and January 2018). At each visit the traps were removed
and new traps were placed, so that a total of 90 Leyer boxes and 135
mesh traps were placed throughout the sampling period. Due to river
flooding and other events, 36% of the Leyer boxes (n=32) and 15% of
the mesh traps (n=20) were lost. During this period, propagules in the
dry season (March 2017 and January 2018) and in the rainy season (June,
August, and November 2017) were collected.
The propagules were identified (to species level or otherwise assigned
to a morphospecies), counted and removed manually. The propagules were
compared with specimens deposited in the Collection of Fruits and Seeds
of the National Herbarium (MEXU), of the Institute of Biology of the
UNAM.
2.3. Data analysis
2.3.1. Temporal variations
To detect differences between seasons, a database was prepared
differentiating the propagules collected in the dry season (propagules
collected in March 2017 and January 2018) and the rainy season
(propagules collected in June, August and November 2017), with which
performed the following analyses in R ver. 4.0.3 (R Core Team, 2020).
Since differences in the number of propagules between seasons, a
standardization of the sampling effort was first carried out by randomly
selecting 52 traps for hydrochory (dry= 26 traps, rainy=26 traps) and 92
traps for other types of dispersal (dry= 46 traps, rainy= 46 traps).
Subsequently, and because the data were not normal, we performed a
Mann-Whitney test to determine differences in the number of propagules
between seasons.
To contrast species richness, a rarefaction analysis was performed using
the iNEXT function (Hsieh et al., 2016) from the iNEXT package.
We calculated the estimated species richness
(q0 ) and its 95% confidence intervals for the
dry and rainy seasons considering the lower coverage that we recorded in
the traps (traps for hydrochory in the rainy season=0.995) (Hsieh et
al., 2016). Finally, to determine changes in species composition, a
permutational multivariate analysis of variance (PERMANOVA) was
performed, applying a Hellinger transformation to the abundance data
using the adonis function from the vegan package in R (Oksanen et
al., 2013). For the PERMANOVA, a distance matrix was used that was
obtained with the Bray Curtis coefficient. To graphically evaluate the
associations in the composition of the propagules dispersed by
hydrochory and by other types of dispersal in the dry and rainy season,
an NMDS was performed with the metaMDS function of the vegan
package (Oksanen et al., 2013).
2.3.2. Longitudinal variations along the streams
To evaluate changes in the abundance and richness of species with the
distance to the origin of the river, a Generalized Linear Mixed Effects
Model (GLMM) with a negative binomial link function was performed using
the glmer.nb function of the lme4 package (Bates, 2010). The
number of propagules/m2 and the number of species
estimated at each site were used as a fixed effect and the stream in
which the sites were established (Yavesia river or Lachatao river) as a
random effect (Fig. 1).
To determine changes in the turnover and nesting of propagule
communities with increasing distance to the origin of the river, values
were obtained for the Simpson dissimilarity index
(βSim ) and nesting between sites near the river
origin (site one: 0.5 km and site two: 0.8 km) and the downstream sites,
in this analysis the sites that were located in the Lachatao stream
(sites six, seven and nine) were excluded because they are located in a
different stream than the rest of the sites. The turnover and nesting
values were obtained using the functions vegdist andnestednodf of vegan package (Oksanen et al., 2013). Subsequently,
a Pearson correlation analysis was carried out to determine if there was
a relationship between the distance to the origin of the river and the
turnover and nesting values.